Cytometry system with interferometric measurement

ABSTRACT

This disclosure concerns methods and apparatus for interferometric spectroscopic measurements of particles with higher signal to noise ratio utilizing an infrared light beam that is split into two beams. At least one beam may be directed through a measurement volume containing a sample including a medium. The two beams may then be recombined and measured by a detector. The phase differential between the two beams may be selected to provide destructive interference when no particle is present in the measurement volume. A sample including medium with a particle is introduced to the measurement volume and the detected change resulting from at least one of resonant mid-infrared absorption, non-resonant mid-infrared absorption, and scattering by the particle may be used to determine a property of the particle. A wide range of properties of particles may be determined, wherein the particles may include living cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following United Statesprovisional application, which is hereby incorporated by reference inits entirety: U.S. Provisional Application No. 61/647,041, entitledSYSTEM AND METHOD FOR HIGH-CONTRAST PARTICLE SPECTROSCOPY, filed May 15,2012.

This application is a continuation-in-part of the following U.S.Non-Provisional patent applications, each of which is herebyincorporated by reference herein in its entirety: U.S. Non-Provisionalpatent application Ser. No. 13/298,148, entitled SYSTEM FOR IDENTIFYINGAND SORTING LIVING CELLS, filed Nov. 16, 2011, and U.S. Non-Provisionalpatent application Ser. No. 13/447,647, entitled CYTOMETRY SYSTEM WITHQUANTUM CASCADE LASER SOURCE, ACOUSTIC DETECTOR, AND MICRO-FLUIDIC CELLHANDLING SYSTEM CONFIGURED FOR INSPECTION OF INDIVIDUAL CELLS, filedApr. 16, 2012.

U.S. Non-Provisional patent application Ser. No. 13/447,647 and U.S.Non-Provisional patent application Ser. No. 13/298,148 each claim thebenefit of the following United States Provisional applications:

U.S. Provisional Application No. 61/456,997, entitled SYSTEM FORIDENTIFYING AND SORTING LIVING CELLS, filed Nov. 16, 2010;

U.S. Provisional Application No. 61/464,775, entitled MICROFLUID SYSTEMFOR LIVE CELL MEASUREMENT, filed Mar. 9, 2011;

U.S. Provisional Application No. 61/516,623, entitled LABEL-FREE FLOWCYTOMETRY SYSTEM, filed Apr. 5, 2011;

U.S. Provisional Application No. 61/519,567, entitled SYSTEM FORHIGH-SPEED LABEL-FREE CELLULAR DNA MEASUREMENT, filed May 25, 2011;

U.S. Provisional Application No. 61/571,051, entitled SYSTEM FORHIGH-SPEED LIQUID PHASE SPECTRAL MEASUREMENTS, filed Jun. 20, 2011

U.S. Provisional Application No. 61/575,799, entitled COMBINEDINSPECTION SYSTEM INCLUDING MID-IR VIBRATIONAL SPECTROSCOPY AND AFLUORESCENT CYTOMETRY FACILITY, filed Aug. 29, 2011; and

U.S. Provisional Application No. 61/628,259, entitled EMULSION-BASEDMIDINFRARED VIBRATIONAL SPECTROSCOPY SYSTEM, filed Oct. 27, 2011.

BACKGROUND

1. Field

This document relates generally to cellular measurements based onmid-infrared absorption measurements and particularly, but not by way oflimitation, cellular measurements based on mid-infrared absorptionmeasurements using mid-infrared laser based architectures for infraredactivated cell sorting (IRACS).

2. Description of the Related Art

Identification, classification and sorting of cells, in particular livecells, is a subject of considerable research and commercial interest.Most recently systems for sorting stem cells have been an area ofparticular focus. For example, methods for separating cancerous fromnon-cancerous cells have been demonstrated. For another example, thereis an established market for cell sorting for gender offspring selectionby identification and selection of X- or Y-bearing spermatozoa.

There is currently no safe and accurate method for cell sorting. Themost advanced technology uses fluorescence-activated cell sorting(FACS), where living cells are incubated in a fluorescent DNA-attachingdye, exposed to a high-intensity, high-energy UV laser beam, and sortedaccording to observed fluorescence. There are two major disadvantages tothis method applied to certain cells, including low accuracy and safetyconcerns. For example, in sperm cell sorting, the FACS process is ableto achieve 88% X-enrichment and only 72% Y-enrichment, even at very lowsort rates (20-30 per second output). High scattering at UV and visiblewavelengths is a major factor. In addition, in sperm cell sorting, theFACS process has been shown to cause chromosomal damage in sperm cellsas a result of the dyes used, and as a result of exposure to highintensity 355 nm laser light.

The use of optical methods to identify and classify cells has manypotential advantages such as speed, selectivity/specificity, and theirnon-invasive nature. As a result, a number of methods have beendemonstrated in which light is used to interrogate cells and determinecritical information. One such method is the use of fluorescent markers,which are chemicals that bind to specific structures or compounds withinthe target cells and are introduced into the mixture of cells. Themixture is subsequently rinsed to remove excess fluorescent markers andthe cells are exposed to intense UV or other short-wavelength radiationin order to “read out” relevant quantities and classify the cell. Thechemical markers provide good specificity. However, these chemicalmarkers may damage or alter the function of the target cells, which isparticularly disadvantageous for live cell sorting. In testing, dyesused as markers for DNA, for example, have resulted in chromosomaldamage. Further, the intense UV or visible light used to read the levelof marker in the cell may damage the cell, in particular, DNA damageresults from exposure to high-energy UV or visible photons. Also,because of the wavelengths used in so called fluorescence activated cellsorting (FACS) systems, quantitative measurements (rather than yes/nomeasurements for a particular antibody) are made very difficult, becauseboth the illuminating wavelength and the emitted fluorescence arescattered and absorbed by cellular components. This means that cellorientation becomes an important factor in accurate measurement, and candramatically reduce the effectiveness of the system. For example, spermcell sorts for X- and Y-carrying sperm, which measure the differentialin DNA between cells, require very specific orientation (only 10% ofcells typically meet the orientation criteria), and still provideaccuracy only in the 70-90% range for humans.

Another method to interrogate cells and determine critical informationis Raman spectroscopy. In Raman Spectroscopy, cells are exposed tointense visible or near infrared (NIR) light. This light is absorbed asa result of molecular bond vibrations within the cellular structure.Secondary emission of photons at slightly different wavelengths occurs,according to Stokes and anti-Stokes energy shifts. Measurement of thesewavelengths allows the chemical composition of the cell to be measured.With Raman spectroscopy, the individual photon energy is generally lowerthan that used for fluorescent markers, however, the net energy absorbedcan be very high and unsafe for live cells. Raman scattering is anextremely weak process: typically only 1 in 10^ 10 incident photons giverise to a Raman-shifted photon, thus requiring long exposure times togenerate sufficient shifted protons for accurate measurement. WhileRaman may not be suitable for high-volume live cell sorting, it can beused in conjunction with other methodologies described herein. Highersensitivity methods such as coherent anti-Stokes Raman scattering (CARS)are being developed which may enable high-throughput screening.

One significant drawback of mid-IR spectroscopy is the strong absorptionby water over much of the “chemical fingerprint” range. This hasstrongly limited the application of Fourier Transform InfraredSpectroscopy (FTIR) techniques to applications involving liquid (andtherefore most live cell applications) where long integration times areallowable—so sufficient light may be gathered to increasesignal-to-noise ratio and therefore the accuracy of the measurement. Thelack of availability of high-intensity, low etendue sources limit thecombination of optical path lengths and short integration times that maybe applied. In addition, because of the extended nature of thetraditional sources used in FTIR, sampling small areas (on the order ofthe size of a single cell) using apertures further decimates the amountof optical power available to the system.

One approach to enabling liquid or solid-state measurements in themid-IR is to use surface techniques. A popular method is the use of anattenuated total reflection (ATR) prism that is positioned directly incontact with the substance of interest (sometimes using high pressure inthe case of solid samples). Mid-IR light penetrates from the prism up toseveral microns into the sample, and attenuates the internal reflectionaccording to its wavelength-dependent absorption characteristics.

Another method which was more recently developed is the use of plasmonicsurfaces which typically consist of conductive layers patterned toproduce resonances at specific wavelengths; at these resonances, thereis coupling into substances places on top of the layer, and again,absorption at a specific wavelength may be measured with good signal.Again, however, the coupling into the substance of interest is veryshallow, typically restricted to microns.

Analysis of particles including biological cells for size, shape andchemical or biochemical content is of great interest in manyapplications including medicine, drug discovery, materials science andmanufacturing, process control, food and water safety, and othermarkets. The characterization of particles by optical scatteringcharacteristics is already widely used in such applications. Forexample, blood counts are performed using scattering-based cytometersthat effectively categorize cells according to size, shape and density.For measurement of biochemical content, however, other methods must beused, or combined with scattering techniques. Most commonly, fluorescentdyes or labels are added to achieve this. This adds significantcomplexity to the measurement process, and limits the applications inwhich particle size, shape, density and biochemical makeup may becharacterized accurately.

One well-known method for assessing biochemical content of condensedphase materials is infrared spectroscopy, usually through the use of aFourier Transform Infrared (FTIR) spectrometer. In FTIR, absorptionspectra of the material under inspection is measured; in the mid-IRrange, molecules have specific absorption bands or “fingerprints”corresponding to molecular bond vibrations. These fingerprints may beused to calculate makeup of a sample, chemical concentrations, and evenmolecular conformations (packing, folding, and other inter- orintra-molecular interactions that are reflected in the bond force/lengthand therefore its characteristic resonant frequency).

One of the problems raised in mid-IR microspectroscopy when particlesare present is that of scattering. First, there is general wavelengthdependence in scattering, with scattering cross-section growing as thewavelength becomes shorter compared to the particle(s) being measured.Second, where particle (or medium) components have strong absorptionfeatures, there is necessarily also (by the Kramers-Kronig relationship)a resonant feature in the real refractive index of the particle ormedium. Since scattering is dependent on both the size of the particleand the refractive index of the particle relative to the medium, thisresults in localized “resonant” scattering. Many groups have developedalgorithms to correct for both the non-resonant and resonant Miescattering effects in FTIR measurements; most of them based on iterativemodels that fit an observed IR absorption spectrum.

Mie scattering is dominant when the particles in the path are on theorder of the interrogating wavelength. The magnitude and angle ofscattering is determined by the size, shape and index of particlesrelative to the medium. Problems are especially prevalent when theparticles or cells being measured have high-index relative to the mediumwhen using mid-IR spectroscopy such as FTIR where scattered light can bemisinterpreted as absorption, and artifacts in the Fourier-invertedspectrum can result. Some of the causes or promoters of this scatteringloss include: 1) Measurement of cells in air medium, rather than in awater medium. This causes additional index mismatch between the mediumand cells, dramatically raising scattering efficiency and angles; 2)Measurement of absorption peaks at high wavenumbers (short wavelengths)where scattering efficiency is higher; 3) Insufficient capture angle onthe instrument, where typically the capture angle on these instrumentsis identical to the input angle, not allowing for light scatteredoutside of the delivered IR beam angle; and 4) Transflection or othersurface-based measurements. These configurations may lead to additionalartifacts in conjunction with Mie scattering effects.

In cytometry techniques, visible or near-infrared wavelengths aretypically used; by measuring the intensity of scattering over a range ofangles the cell size may be estimated. For example, some modern bloodcount equipment uses this method to approximate blood cell size andshape to generate a detailed blood count. However, the scatteringdistribution resulting from laser illumination at these wavelengths isdependent on many factors, including cell shape, orientation, density,and chemical composition. It is not possible to determine chemicalcomposition at these wavelengths, and therefore, to eliminate thisfactor which affects scattering pattern and therefore volume estimate.

The ability to measure particles or cells suspended in liquid, eitherindividually or in aggregate, using mid-IR spectroscopic methods hassignificant implications in a number of applications, both in thebiomedical market and in other markets. Ideally, an optical method couldbe devised that would estimate the volume of the particle that waschemically distinct from the medium surrounding (and in some casespermeating) it. However, one of the challenges of mid-IR spectroscopy onparticles or cells, particularly where high throughput is required, isgetting sufficient contrast as a particle passes through the measurementvolume. This is particularly acute when multiple wavelengths are usedsimultaneously (for example, modulated at different carrierfrequencies), and each is only absorbed or scattered in a small fractionby the particle(s). For cells suspended in water, the significantabsorption bands associated with water in the mid-IR pose a challenge.

In many applications where small particles are measured, it is useful tomeasure the volume of the particles. A related and often more importantmeasurement is the content in the particle excluding its medium. Forexample, when measuring biological cells, the non-water volume of a cellcan be a strong marker for cell phenotype, and may in addition containsignificant information on the status of the cell (for example, if it isactively dividing). Multiple methods for estimating cell volume havebeen devised.

One device, which approximates volume, is a Coulter Counter, which usesa voltage potential over a channel filled with conductive medium thoughwhich biological cells flow; as the cells pass through the channel, theyblock electrical current, with the reduction in current indicative ofcell volume. This device may be used, for example, to differentiate redfrom white blood cells and rapidly generate a blood count. CoulterCounters are used outside of biology as well in applications such aspaint, ceramics, glass and food manufacturing where particle sizing (anddistribution of sizing) is of high importance.

This method, while very useful for measuring particle volume, isdependent on the precise composition of the particle or cell, includingwhether its membrane is electrically insulating and on the conductivityof liquid contained inside the cell. For estimating total non-water (ormore generally non-medium) volume, it would be preferable to eliminatethis dependency. Additionally, the requirement for an electricallyconductive medium places limits on the materials and particles that maybe measured with a resistance-based method such as the Coulter Counter.While capacitive methods have been employed as well, these are highlysensitive to particle position and other environmental factors.

In the quest to provide accurate measurements of true weight of aparticle, or more specifically a biological cell, one group (Manalis etal at MIT) have gone so far as to build an ultra-sensitive “scale” basedon a microfluidic channel on a microfabricated cantilever, though whichbiological cells are flowed. The characteristic resonant frequency ofthe cantilever is shifted as each cell passes through the tip of thecantilever, allowing measurement of cell mass. This device has beenproposed as a method to repetitively measure individual cell massesthrough the course of a treatment (for example, as a drug or othertreatment is applied to a population of cancer cells). While this methodis novel and potentially highly accurate, it is highly complex (requiressignificant difficult fabrication, calibration, compensation) andpotentially suffers from low throughput (flow rates must be kept low toprovide an accurate measurement and prevent rapid clogging).

Both of the aforementioned methods (Coulter Counter and cantilever“scale”) also have the disadvantage that they may be difficult tointegrate with other measurement techniques. Specifically, in biomedicalapplications where biological cells are measured, much additionalcellular characterization is done optically, by measurement of scatteredlight and/or fluorescence induced in the cell or chemical dyes/labelsthat have been added to stain or mark the cell. Ideally, a method formeasuring non-medium volume of a particle or cell could be integratedseamlessly with these other measurements to provide an integratedmeasurement. In other words, an optical method for measuring non-mediumvolume would be strongly preferred. This would additionally not requirea medium that is electrically conductive.

Thus there remains a need for techniques to identify and measureparticles or cells that provide accurate results and are usable onliving cells.

SUMMARY

The present invention provides a cytometry system for measuringcharacteristics of a cell or particle including chemical composition orphysical characteristics. The cytometry system includes a handlingsystem that presents a cell or particle to a laser light source formeasurement by transmission or scattering, wherein the laser can be aquantum cascade laser (QCL). Infrared light is used to reduce the lightenergy so that living cells can be measured and also to reducescattering from cells or particles that are on the order of a micron insize. Visible light sources can be included to aid in the identificationof the location of the cell or particle as it moves through the system.The laser light source can provide multiple wavelengths of light formeasurement. Measurements can be differential either by measuringmultiple positions or by performing multiple measurements with andwithout cells or particles in a measurement volume. Cells or particlesare presented to the cytometry system in a medium such as a liquid forimproved handling in a flow. Quantum cascade lasers provide highintensity with multiple wavelengths to transmit through the flow forimproved measurement capability.

In an embodiment, a method for measuring a particle in a medium includesproviding an infrared light source with one wavelength that correspondsto a non-resonant vibrational condition for a material of the particleand another wavelength that corresponds to a resonant vibrationalcondition for a material of the particle. A measurement volume is alsoprovided for conducting measurements of characteristics of the particlein the medium. The measurement volume is illuminated by the infraredlight source so the infrared light passes over the particle in themedium. Light that is scattered by the particle is then detected andanalyzed. Size or shape of the particle is determined in correspondenceto the detected light associated with the non-resonant vibrationalcondition, while chemical makeup of the particle is determined incorrespondence to the detected light associated with the resonantvibrational condition. This method can also be used to measure aplurality of particles in a medium and used to sort individual particlesbased on their respective determined characteristics. The determinedcharacteristics can include one or more of the following: size, shape,refractive index, density, DNA content, protein content, lipid content,sugar content, RNA content, molecular structure, crystal structure, andchemical makeup. The detected changes in transmitted or scattered lightcan include changes in intensity or changes in angle.

In another embodiment, a method is provided for measuring a chemicalcomposition of a liquid medium by illuminating a particle in the mediumwhere the particle is selected to have characteristics that enable aspecific chemical makeup of the medium to be determined. An infraredlight source provides light to illuminate the medium and particle as itflows through a measurement volume. Light that is scattered by theparticle as it passes through the measurement volume is detected. Thechemical makeup of the medium is determined in correspondence to thedetected light.

In an embodiment a method for interferometric spectroscopic measurementsof particles with favorable signal to noise is provided using amid-infrared light. The mid-infrared light is split into two beams andat least one of the beams is directed to pass through a measurementvolume containing a sample. The two beams are then combined to provide arecombined beam, which is measured by at least one mid-infrareddetector. Wherein the relative phase delay between the two beams iscreated so the beams destructively interfere when they are recombined.When a sample is introduced to the measurement volume that includesmedium with at least one particle, changes in the scattering orintensity of the recombined beam are detected and used to determine aproperty of the particle. The determined characteristics can include oneor more of the following: a chemical composition, a physicalcharacteristic, size, shape, refractive index, density, DNA content,protein content, lipid content, sugar content, RNA content, molecularstructure, crystal structure, and chemical makeup. The detected changesin transmitted or scattered light can include changes in intensity orchanges in angle. The particle may be at least one of a biological cell,a tissue sample, a bacterium, a blood sample, and an embryo. The mediumis a liquid and the particle is an emulsion. The method may furtherincluding passing at least one of the beams through an attenuator andadjusting the attenuator to decrease the intensity of the beam. At leastone of the beams passes through a low-pass or high-pass spatial filterbefore recombining. The light source is a laser, which may be a quantumcascade laser.

In another embodiment, another method for interferometric spectroscopicmeasurements of particles with favorable signal to noise is providedutilizing a mid-infrared light source with a light beam that is splitinto two beams. At least one beam is directed through a measurementvolume containing a sample including a medium. The two beams are thenrecombined and measured by a mid-infrared detector. The phasedifferential between the two beams is created to provide destructiveinterference if no particle is present in the measurement volume. Asample comprising medium with a particle is introduced to themeasurement volume and the detected change resulting from resonant ornon-resonant mid-infrared absorption or scattering by the particle isused to determine a property of the particle. The determinedcharacteristics can include one or more of the following: a chemicalcomposition, a physical characteristic, size, shape, refractive index,density, DNA content, protein content, lipid content, sugar content, RNAcontent, molecular structure, crystal structure, and chemical makeup.The detected changes in transmitted or scattered light can includechanges in intensity or changes in angle. The particle may be at leastone of a biological cell, a tissue sample, a bacterium, a blood sample,and an embryo. The medium is a liquid and the particle is an emulsion.The method may further including passing at least one of the beamsthrough an attenuator and adjusting the attenuator to decrease theintensity of the beam. At least one of the beams passes through alow-pass or high-pass spatial filter before recombining. The lightsource is a laser, which may be a quantum cascade laser.

In an embodiment, an interferometric spectroscopic apparatus is providedwhich provides favorable signal to noise measurements of particlesutilizing a mid-infrared light source providing one or more wavelengthsin a light beam applied to a measurement volume containing a sample.Wherein a first beam splitter splits the light beam into two beams andoptics direct at least one of the beams through the measurement volume.An adjustable phase delay apparatus on one of the beams is configured toresult in destructive interference between the two beams when the sampleis comprised of medium without particles. A combiner combines the twobeams to provide a recombined beam, which is detected by a mid-infrareddetector. Changes in transmitted or scattered light in the recombinedbeam that occur when a sample comprised of medium and one more particlesis introduced to the measurement volume are used to determine a propertyof the particle. A processor may determine the property by comparing thechanges to a series of known particle properties. The light source is atleast one of a laser and a synchrotron. The laser is a quantum cascadelaser. The detector is at least one of a mid-infrared focal plane array,a mid-infrared image sensor, a scanning detector, and a detector with aspatial light modulator. The detector measures intensity as a functionof scattering angle and wavelength. The apparatus further includes anattenuator configured such that at least one of the beams is able to bepassed through it to change the intensity of the beam and wherein saidattenuator is capable of changing the intensity of the beam when thebeam is passed through it such that the intensity of the recombined beamwhen the sample does not comprise a particle is reduced. The apparatusfurther includes a low-pass or high-pass spatial filter, both configuredsuch that at least one of the beams is able to be passed through itbefore recombining. The phase delay apparatus includes an adjustablemirror assembly. The phase delay apparatus includes a phase delay block.

In a further embodiment, a micro-spectroscopic system is provided inwhich an image of a sample as well as chemical makeup information isprovided. The system includes a quantum cascade laser light source thatprovides one or more mid-infrared wavelengths of light, wherein at leastone of the wavelengths corresponds to a resonant condition related to achemical makeup of a portion of the sample. First optics are provided todirect laser light to illuminate an area of the sample in a holder.Second optics direct scattered light produced by laser light passingthrough the sample to an imaging detector where the light is detected.The chemical makeup and structure of the sample is determined based onanalysis of the detected light. An image of the sample along withinformation related to a chemical makeup and structure of the sample isprovided in correspondence to the detected light. The imaging detectorcan be a focal plane array, an image sensor, a scanning detector or acoded aperture imaging system. To reduce coherence in the laser lightand thereby improve characteristic of the image of the sample that isprovided, the first optics can include spatially defined time varyingpath lengths.

In another embodiment, a method for measuring the volume of a particlein a medium is provided that uses a light source with one or morewavelengths where the medium is absorptive and the particle is lessabsorptive. The medium is presented for measurement in a constantthickness that contains the particle. A light beam from the light sourceis transmitted through the medium containing the particle. Changes inthe transmitted light are detected when the particle passes through thelight beam. The volume of the particle is determined in correspondenceto the detected changes in transmitted light. The particle can be abiological cell, a tissue sample, a bacterium, an embryo and a bloodsample. The medium can be a liquid or a solid. When the medium is aliquid, the particle can be an emulsion. The method can be used to sorta plurality of particles based on their respective determined volumes.The wavelengths can include infrared and visible wavelengths.Fluorescence from the particles can also be detected and used to helpdetermine a volume of the particle.

A further embodiment provides a method for measuring the concentrationof particles in a medium when the size of the particles is known. Alight source is utilized with one or more wavelengths where the mediumis absorptive and the particles are less absorptive. Constant thicknesssamples of the medium are provided with and without particles. A lightbeam from the light source is transmitted through the samples anddetected. Changes in the transmitted light are detected between thesamples with and without particles. The concentration of the particles,in the sample with particles, is determined in correspondence to thedetected changes in transmitted light. The particles can includebiological cells, tissue samples, bacteria, embryos, or blood samples.The wavelengths can include infrared and visible wavelengths.

Yet another embodiment is a method for measuring the volume of aparticle in a medium using an infrared light source with one or morewavelengths, where the refractive index of the medium varies sharply andthe refractive index of the particle is relatively constant. Theparticle is provided for measurement in a medium of constant thickness.A light beam from the light source is transmitted through the mediumcontaining the particle. Changes in scattered light are detected whenthe particle passes through the light beam. The volume of the particleis determined in correspondence to the detected changes in scatteredlight. In an alternate embodiment, the light source has two or morewavelengths wherein the refractive index of the medium varies sharply atone of the wavelengths and the refractive index of the particle isrelatively constant at another one of the wavelengths. Changes inscattered light are then detected at the two or more wavelengths whenthe particle passes through the light beam and the volume of theparticle is determined in correspondence to the detected changes inscattered light at the two or more wavelengths.

In embodiments, the system includes a mid-infrared laser light sourcesuch as a quantum cascade laser for measuring a cell or particle usinglaser light sources that provide multiple spots. Multiple laser lightsources can provide the multiple spots or alternately, optics can beincluded to provide multiple spots from one laser light source. Ahandling system delivers the cell or particle so that it passes throughthe multiple spots. A light measurement system measures the effect ofthe cell or particle passing through the spots. Characteristics of thecell or particle are determined in correspondence to the measuredeffect.

In another embodiment, a method for measuring a particle with improvedsignal to noise is provided that utilizes a mid-infrared light sourcewith one or more wavelengths in a patterned beam. A sample is presentedfor measurement comprised of a particle in a medium so that the particlepasses through a measurement volume. The patterned beam illuminates themeasurement volume. Changes in transmitted or scattered light aredetected as the particle passes through the measurement volume.Characteristics of the particle or medium are determined based on thedetected changes. The patterned beam can include a series of spots, aseries of lines or a series of ellipses. The determined characteristicscan include one or more of the following: size, shape, refractive index,density, DNA content, protein content, lipid content, sugar content, RNAcontent, molecular structure, crystal structure, and chemical makeup.The detected changes in transmitted or scattered light can includechanges in intensity or changes in angle.

Yet another embodiment provides an improved spectroscopic apparatus forproviding measurements of particles with higher signal to noise. Amid-infrared light source provides one or more wavelengths in a beam.First optics provide the beam as a patterned beam that illuminates ameasurement volume. A flow controller causes particles in a medium toflow through the measurement volume. Second optics direct transmitted orscattered light from the measurement volume to a detector where changesin the transmitted or scattered light are detected as the particles inthe medium flow through the measurement volume. A processor compares thedetected changes to known characteristics of particles to determinecharacteristics of the particles. The first optics can include awavelength dispersive element, a diffraction grating, a patterned phasegrating, a slit, a slit array or a spatial modulator. The patterned beamcan include a series of spots, a series of lines or a series of ellipsesin a two or three dimensional pattern. The determined characteristics ofthe particle can include at least one of the following: size, shape,refractive index, density, DNA content, protein content, lipid content,sugar content, RNA content, molecular structure, crystal structure, andchemical makeup. The light source can be a broadband Fabry-Perot quantumcascade laser. The detector can be a single detector, an array ofdetectors, a focal plane array or an image sensor.

In further embodiments, the cytometry system may be used to present asingle sperm cell to at least one laser source configured to deliverlight to the sperm cell in order to induce bond vibrations in the spermcell DNA, and detecting the signature of the bond vibrations. The bondvibration signature is then used to calculate a DNA content carried bythe sperm cell which is used to identify the sperm cell as carrying anX-chromosome or Y-chromosome. Another system and method may includeflowing cells past at least one laser source one-by-one using a fluidhandling system, delivering laser light to a single cell to induceresonant mid-IR absorption by one or more analytes of the cell, anddetecting, using a mid-infrared detection facility, the transmittedmid-infrared wavelength light, wherein the transmitted mid-infraredwavelength light is used to identify a cell characteristic.

These and other systems, methods, objects, features, and advantages ofthe present invention will be apparent to those skilled in the art fromthe following detailed description of the preferred embodiment and thedrawings. All documents mentioned herein are hereby incorporated intheir entirety by reference.

BRIEF DESCRIPTION OF THE FIGURES

The invention and the following detailed description of certainembodiments thereof may be understood by reference to the followingfigures:

FIG. 1 illustrates the present invention built in a form similar to aflow cytometer.

FIG. 2 shows a potential configuration of laser source to interrogate asample stream, in either flow cytometer configuration as shown in FIG.1.

FIG. 3 illustrates a simplified example of mid-infrared spectra for aflow such as those described in FIG. 1 and FIG. 2.

FIG. 4 shows an example configuration of a system interrogating cells ina flow, which is shown in cross-section.

FIG. 5 shows an embodiment of the present invention, in which the flowand cells 120 and 122 are measured from multiple angles.

FIG. 6 illustrates simplified detector signal, corresponding to thesample spectra shown in FIG. 3.

FIG. 7 shows another embodiment of the present invention, where cellsare measured in a dry state.

FIG. 8 shows the application of the present invention embodied using amicrofluidic-type cell sorting system.

FIG. 9 shows a portion of a very basic embodiment of the presentinvention which is a microfluidic system for live cell measurements.

FIG. 10a shows detail of an embodiment of a measurement region in amicrofluidic channel.

FIG. 10b shows the same example as 10 a in cross-section.

FIG. 10c shows a cross-section of an alternative embodiment that may usea reflective measurement for the mid-IR light.

FIG. 11 shows an embodiment of the invention where the microfluidics andQCL-based spectral measurement system may be combined with a moreconventional fluorescence-based measurement system.

FIG. 12 shows an exemplary embodiment of the present invention where themicrofluidic subsystem includes a cell-sorting fluidic switch.

FIG. 13a shows an alternative microfluidic-based embodiment of thepresent invention, where a series of microwells may be integrated into amicrofluidic flow channel/chamber.

FIG. 13b shows how the wells may be then scanned using mid-IR light fromone or more QCLs, the scanning may be accomplished by translating themicrofluidic chip, or the laser leading mechanism.

FIG. 14 depicts another embodiment in which the microfluidic chamber maybe 2-dimensional.

FIG. 15 shows an alternate embodiment of the mid-IR optics combined witha microfluidic channel.

FIG. 16 shows a system for the growth and purification of cells based onthe present invention.

FIGS. 17a and 17b shows two potential configurations for QCLs and mid-IRdetectors in the present invention.

FIG. 18 shows an embodiment of the present invention where it may beused to sort live sperm cells for the purpose of pre-fertilization sexselection.

FIG. 19a shows a cross-section of a fluid stream oriented to carry cellsthrough a measurement volume such that flow is into or out of plane ofpaper in this case.

FIG. 19b shows the same configuration with two example cells in theflow.

FIG. 20a illustrates one configuration of the present invention thatminimizes the effect of cell position in the flow that results fromwater absorption.

FIG. 20b illustrates the rectangular channel with two hypothetical cellpositions in the measurement volume, a well-centered cell and anoff-axis cell.

FIG. 21 shows an embodiment of the present invention implemented in astandard flow cytometry system, in this case fitted for mid-IR cellmeasurements based on QCLs.

FIG. 22 shows the present invention embodied in an architecture thatallows cell position in a flow to be measured accurately, in order tocompensate for position-dependent variations in mid-IR absorptionsignal.

FIG. 23a shows a sheath flow surrounding the core flow being illuminatedusing mid-IR light from QCL(s).

FIG. 23b shows the same flow with a cell in the measurement volume.

FIG. 24 depicts another embodiment of the present invention in whichmultiple mid-IR beams may be used to interrogate the measurement volume.

FIG. 25a shows a tool that may accept microfluidic chips that may bepre-loaded by user with the cell sample to be measured and/or sorted.

FIG. 25b shows another configuration of the tool described herein.

FIG. 26a shows a configuration where DNA vs. cell count is displayed ina histogram format.

FIG. 26b shows a configuration where an additional parameter besides DNAis used to classify cells.

FIG. 27 shows an example of a single-unit disposable measurement unit,consisting of a microfluidic chip with plastic carrier that may be usedin a system.

FIG. 28 shows an alternative configuration, where a potentially plasticcarrier may be used together with one or more microfluidic chips, wherethe chip may be a separate piece from the carrier.

FIG. 29a shows an example format for a microfluidic chip used in ameasurement-only application.

FIG. 29b shows an example format for a microfluidic chip for use insorting cells.

FIG. 30a shows an example format for a microfluidic chip with aconfiguration where a diluting/sheath fluid is used together with thesample in order to provide a centered flow in the measurement channels.

FIG. 30b shows an example format for a microfluidic chip in aconfiguration where cells are switched, based on the vibrationalspectroscopy measurement, using an electric field at the switch point.

FIG. 31 shows the example construction of a microfluidic chip for use inthe present invention.

FIG. 32 shows the detail of an example embodiment of a microfluidicchannel including a measurement volume and a pressure-actuated cellswitch.

FIG. 33 illustrates an embodiment of the optical interrogation systemused in the present invention.

FIG. 34 shows an example of the present invention using coherentanti-stokes Raman spectroscopy (CARS) to measure vibrational bondfingerprints in cells at high speed.

FIG. 35 shows a configuration of a QCL with components to reducecoherence length, so as to minimize spatial dependence in the readout ofcell spectral measurements.

FIG. 36 shows an example of the display/user interface when the tool maybe used for cell sorting.

FIG. 37 shows a sample embodiment of the present invention that mayinclude elements to minimize spot size on the sample, and to rejectscattered light from the sample.

FIG. 38a shows an attenuated total reflectance ATR configuration oftenused by others to measure liquids or solids using Fourier TransformInfrared (FTIR) spectroscopy in the mid-IR.

FIG. 38b shows a configuration used by others that uses plasmonic layers(patterned metal conductive layers) to enhance absorption.

FIG. 38c shows a true transmission architecture used by others that mayhave been used for mid-IR measurements.

FIG. 39 shows an example embodiment of a microfluidic channel describedin the present invention.

FIG. 40a shows the electric field for a microfluidic gap which is aneven quarter wave multiple of the interrogating wavelength.

FIG. 40b shows the optical intensity for the microfluidic gap of FIG. 40a.

FIG. 40c shows a gap not resonant at the interrogating wavelength(s)which ensures more uniform sampling of the contents of the fluidicchannel.

FIG. 41 shows an embodiment of the present invention based on aVernier-tuned external cavity quantum cascade laser.

FIG. 42 further illustrates the Vernier tuning mechanism proposed forhigh-speed mid-IR liquid spectroscopy as part of the present invention.

FIG. 43a shows a configuration where an absorption spectrum may bemeasured at an absorption peak of interest, with three points beingmeasured on the peak.

FIG. 43b shows a more minimal configuration than that of FIG. 43a ,wherein only a peak absorption wavelength and a reference wavelength aresampled.

FIG. 44 shows another example embodiment of the present invention.

FIG. 45a shows the absorption of the particle and the medium in which itis measured, both as a function of optical frequency.

FIG. 45b shows the derived real refractive index of the particle and themedium.

FIG. 46 shows a generalized system diagram for measurement of mid-IRabsorption of a particle of cell, including but not limited to livingcells.

FIG. 47 shows one approach to remedying the scattering problem.

FIG. 48 shows an alternative architecture where scattered light ismeasured directly.

FIG. 49 shows an embodiment that has the potential to use QCLs tomeasure particle size and chemical concentration through scattered lightonly.

FIG. 50a depicts shows a simple flow architecture where the fluid to bemeasured flows through a channel.

FIG. 50b depicts a fluid-within-fluid flow.

FIG. 50c depicts an example of a flow where the core has been brokeninto droplets.

FIG. 51a shows an example of scattering efficiency (Qs) of a volume suchas a droplet, as a function of wavelength (lambda).

FIG. 51b shows scattering efficiency as a function of wavelength

FIG. 52a shows a fluidic configuration where a droplet or flow isconfined within a 2D channel or manifold.

FIG. 52b shows a fluidic system where droplets or a core flow iscentered within a larger, 3D core flow.

FIGS. 53a-c show a representative example of a system measuring dropletsor flows using QCL-originated mid-IR beams.

FIG. 54a shows a case where a biological cell (inner circle) iscontained within a droplet, which is contained within an emulsion.

FIG. 54b shows a different technique, illustrate here through the use ofa droplet in an emulsion.

FIG. 55 shows an example of a droplet-based system where dropletscontain biological cells.

FIG. 56 shows a configuration where a solid sample with very sparseparticles of interest.

FIGS. 57a-c show flow and particle configurations that could be used toenhance resonant optical interference measurements in the mid-IR.

FIG. 58 shows one method by which cells or particles could be measuredand sorted.

FIG. 59a is a graph of refractive index vs wavelength for a particle anda medium that do not have a resonant vibration peak.

FIG. 59b is a graph of scattering vs wavelength for two particles (P1,P2) of the same refractive index and different sizes in a medium.

FIG. 59c is a graph of refractive index vs wavelength for a particle anda medium that have a resonant vibration peak.

FIG. 59d is a graph of scattering vs wavelength for two particles (P1,P2) of the same refractive index and different sizes in a medium wherethere is a resonant peak.

FIG. 60 is a schematic drawing of a vibrational spectroscopy system witha beam block after the light passes through the sample.

FIG. 61 is a schematic drawing of a vibrational spectroscopy system witha beam block before the light passes through the sample.

FIG. 62 is a schematic drawing of a vibrational spectroscopy system witha lens to focus the unscattered light onto a beam block and another lensto focus the scattered light onto the detector.

FIG. 63 is a schematic drawing of a system that simultaneously capturesscattering angle and wavelength vs intensity.

FIG. 64 is a schematic drawing of an interferometer spectroscopy systemwith a variable delay block.

FIG. 64a is a schematic drawing of an interferometer spectroscopy systemwith a variable path length phase delay.

FIG. 65 is a schematic drawing of a system including a diffractiveelement to provide multiple spots of light to the measurement volume.

FIG. 66 is a schematic drawing of a system including a light source withmultiple wavelengths and a diffractive element to spread the wavelengthsspatially over the measurement volume.

FIGS. 67a-c are illustrations of multiple spots of light across themeasurement volume.

FIG. 68 is a schematic drawing of different spectrums associated withdetermining the non-water components of cells.

FIG. 69 is a schematic drawing of a system including a spatial lightmodulator.

FIG. 70 is an illustration of a particle moving laterally through aseries of spots of light from two different wavelength light sources.

FIG. 71 is a flowchart for a method of the disclosure.

DETAILED DESCRIPTION

The disclosure herein presents a novel approach to cytometric cellularmeasurements based on mid-infrared absorption measurements includingvibrational spectroscopy systems. Mid-IR lasers have the potential tofocus sufficient energy onto a particle or a single cell to make anaccurate, high-speed measurement. Recently a number of quantum cascadelasers (QCL's) been commercialized by multiple vendors including AlpesLasers SA, Neuchatel, Switzerland (http://www.alpeslasers.ch/) thatprovide mid-infrared laser light. They are built using the sameprocesses and packaging as high-volume telecom lasers. QCLs offerseveral advantages over traditional mid-IR sources, such as deliveringvery high spectral power density, delivering very high spatial orangular power density, among other advantages. This allows QCLs to put10,000,000× more effective mid-IR power onto a single cell thantraditional mid-IR sources. This disclosure seeks to describe certainmethods and systems enabled by mid-IR lasers including QCLs, such aslabel-free detection without dyes or labels that can alter or damagecells, measurements using mid-IR illumination 25× less energetic thanthat used in FACS, eliminating photon damage, and high-throughput (>1000cells/second) capability.

Embodiments of the present disclosure include an Infrared-Activated CellSorting (IRACS). The steps in an IRACS system can include preparing thecell sample (such as by centrifugation, etc.); for each cell flowingthrough: illuminating the cell with mid-IR laser(s), measuring thetransmission of mid-IR wavelength(s), and sorting cells by transmissionor scattering levels. The key “input” parameters of the IRACS systemmodel may be: cells per second entering the measurement volume; andspacing between cells (“duty cycle”). The input parameters may determinethe measurement duration or integration time. Throughout thisSpecification, UV light may be in the range of 10 nm to 400 nm, NIRlight may be in the range of 0.75-1.7 μm, and visible light may be inthe range of 390 to 750 nm.

Most of the risks at the micro optical level may be addressed by carefuldesign using existing techniques and materials. In this manner many ofthe mid-IR measurement issues may be avoided. The IRACS system mayinclude a microfluidic channel architecture with one or more of thefollowing features. One feature may be top and bottom mid-IRtransmitting windows forming a channel. Potential materials include: Si,Ge, ZnSe, certain polymers. Si, ZnSe (or similar), polymers may becompatible with visible, NIR, or SWIR (500-1600 nm) opticalinterrogation or manipulation, if desired. ZnSe or similar may be usedto permit visible light viewing, interrogation and possible manipulation(laser tweezers, laser-based cell disabling/destruction). Anotherfeature may be antireflection coatings applied to both sides of eachwindow, such as AR coatings designed for air externally, waterinternally. Another feature may be a channel depth tuned to be a gapthat is out of resonance at the mid-IR wavelength(s). For example, achannel depth of approximately 20 microns may be used. Another featuremay be tilting, or wedge-shaped windows to further reduce effects ofreflections. Another feature may be a channel width that exceeds thespot size of the laser so small shifts in the channel with respect tothe beam don't cause false signals.

Further, in the present disclosure, several aspects of particularmeasurements which greatly mitigate the effect of Mie scattering by theinterrogated cell may be: 1) The cell is measured in a water medium (asopposed to a dried cell on a window, in air). This may significantlyreduce the index contrast between the medium and cell. The estimatedreal cellular refractive index may be in the range of n_(cell)=1.27-1.30vs n_(H2O)=1.25 at 1100 cm-1, for a cell/medium ratio of 1.02-1.04, anda nuclear refractive index higher than that; 2) DNA signatures are atthe low-wavenumber (long wavelength) end of the vibrational fingerprintregion. Scattering falls off with longer wavelength, so relatively lesseffect is experienced; and 3) Light is captured over a large angle usinga high NA lens; this means even light scattered over moderate angles (10degrees) may be captured and relayed to the detector.

Regarding the issue of cell orientation, scattering is estimated basedon an equivalent spherical volume, and the computer scatteringefficiency is multiplied by the actual cell cross-section that isdependent on cell presentation angle. This may ignore the fact that whencell cross-section is large, path length is relatively short, whichwould in turn reduce scattering effects (since scattering is a result ofthe phase shift and extinction on passage through the sample vs.surrounding medium). To further reduce the effect of cell orientation,the present invention may consider the following approaches: 1) Removingoutliers from the distribution (which may be caused in large part by theorientation issues); 2) Using a measurement wavelength that may not beat the peak absorption level for DNA, or using a less strongly-absorbingDNA band. A high absorption coefficient results in both largerscattering, and also in more orientation/shape dependence instraight-through absorption. At low absorption, path length may bettercompensate for cross-section changes. At high absorption this may beless true; and 3) Using a shorter wavelength (possibly NIR) to moreprecisely determine cell cross-section/orientation, and compensating foror rejecting certain orientations.

Further, to reduce or compensate for the effects of scattering, thepresent disclosure may employ a plurality of solutions such aswavelength optimization, beam angle and capture angle optimization,scatter detection and compensation, scatter based measurement, and thelike.

In an embodiment, the invention uses quantum cascade laser (QCL)components to directly measure absorption spectra of living cells forclassification. Some areas where QCLs are used may includedifferentiating gender in sperm cells based on differences in X- andY-chromosome mass, separation of stem cells from differentiated cellsbased on DNA/RNA mass change, differentiation of healthy and diseasedcells, identification of cell phase of life, and the like.

In an embodiment of a DNA measurement system, the system utilizesoptical absorption into molecular vibration modes specific to the DNAbackbone to accurately measure DNA content of cells as they pass anoptical illumination and readout system in a fluid stream. The systemrequires no staining or labels, or associated incubation process. Thesystem may present a histogram showing cell count vs. DNA quantity percell, and may optionally allow the user to sort out cells with certainquantities of DNA. The system may include reference measures toestablish cell size (such as a Coulter-type electrical impedancemeasurement, or an optical scattering measurement), or other cellularcomponents (such as protein content), which may further help distinguishcell types, and allow identification of cell agglomerates so as toremove them from the data or the sorted stream.

In various embodiments, one or more lasers may be used to measuretransmission at one or more wavelengths through a living cell. Anabsorption by a cell of the one or more wavelengths may indicateconcentrations or mass of constituent components within the cell.Further an absolute or relative level of absorption may be used toclassify or identify the cell type or state. The absorption lines maycorrespond to vibrational modes of molecular bonds in target molecules.

QCLs may be able to directly emit coherent radiation in the mid-infraredrange, covering at least the 3-15 micron wavelength range, such as 3-4microns. The wavelength may coincide with the regions for infraredspectroscopy, and with the wavelengths used in many of the previousspectroscopic work on cells. The potential advantages of usingmid-infrared light to interrogate cells for classification or sortingare numerous, however. They include very specific signatures, low photonenergy, and direct measurement that potentially allow combinations oflow optical power, fast measurement, and/or high precision measurement.Mid-infrared is an important range, since in this range, molecularvibrations may be measured directly through the use of absorptionmeasurements. Essentially the same vibration signals that are measuredusing Raman spectroscopy are measured. Some major advantages of themid-infrared range include that the absorption rate is much higher thanin Raman, resulting in a significantly higher signal per input photon.Additionally, the photon energy used is extremely low compared tovisible-light or even near-infrared measurements; this means no damageto cells or their components from ionization or two-photon absorptionprocesses. Finally, molecular fingerprints in this range have beenextensively characterized for decades using Fourier transform infrared(FTIR) spectroscopy. As opposed to FTIR, which typically uses a “globar”(glowing filament) source which provides very low spectral and arealpower density, QCLs provide much more power on target and on wavelength,allowing much higher signal-to-noise (SNR) ratio and/or throughput.

QCLs may be manufactured in a number of varieties, including Fabry-Perotand Distributed Feedback (DFB) designs, as well as external cavity (EC)designs where wavelength may be set using external devices, and may bebroadly tunable. Advantages of QCLs as a source for mid-infrared lightfor use in microspectroscopy of living cells may include ability todeliver a large amount of optical power in a narrow spectral band,ability to focus mid-infrared light onto a small spot, ability toproduce significant power levels, ability to source QCLs in smallpackages, and the like.

In an embodiment, mid-infrared QCLs that may be used in the presentinvention generally target but are not restricted to the molecular“fingerprint region” of 6-12 microns. In some embodiments, the presentinvention may be applied to the problem of sorting living cells at highspeed in a label-free manner, using relatively small amounts of opticalpower, and using very low-energy photons such as wavelengths around the1000 cm^-1 range to prevent damaging the cells. In various otherembodiments, multiple QCL wavelengths may be used to measure relativeconcentrations of one or more substances within cells, and establish abaseline measurement for the main measurement being performed. The QCLwavelengths may be supplemented by visible, near-infrared or otherwavelength measurements to provide reference information such as celllocation, shape, orientation, scattering, etc. In an embodiment,multiple QCL wavelengths may be generated through the use of multiplediscrete components, single components generating multiple discretewavelengths, broadband QCLs in addition to a filtering technique,tunable QCL components and the like. A mid-infrared source, such as thequantum cascade laser (QCL)—may be integrated with microfluidic systemsfor cell transport, presentation to the measurement system, andoptionally, sorting into specific populations.

Microfluidic systems are in broad use in biomedical applications,including high-volume commercial applications. Their fabrication and usemay be well-known. Microfluidics may be used to combine, measure, sort,and filter biochemical samples. In some embodiments, the combination ofmicrofluidics with QCLs and mid-IR detectors to achieve accuratemeasurements of live cells may be described. Microfluidics may enablehigher accuracy in such a system. Mid-IR light can be absorbed verystrongly by water. Such a system with small path length (through liquid)may be highly desirable for systems requiring high throughput, lowintegration time, or very high signal-to-noise ratio. Furthermore, itmakes a repeatable, constant path length desirable in order to eliminatefluctuations due to water stream diameter. Microfluidic devices orcircuits, which are fabricated using semiconductor-like processes, mayhave the potential to provide such repeatability.

In addition, microfluidic devices enable closed-loop, compact systemsthat may be more appropriate for systems produced and used in highvolume, which may be one goal of the present invention. Microfluidiccomponents may be fabricated at low cost, and may therefore bedisposable or recyclable and may offer a low-cost way of maintaining aclean system without run-to-run or patient-to-patient contamination.

In some embodiments, the combination of a label-free cellcharacterization system based on mid-IR QCLs may enablesystems-on-a-chip with cell culturing, filtering, detecting, and sortingon a single chip, thereby minimizing inputs and outputs. The ability touse QCLs rather than fluorescent, magnetic or other labels (that must beattached to cells through specific, sometimes laborious procedures thatmay ultimately affect cell viability) multiplies the number ofoperations that may be performed on-chip for specific biomedical or evenindustrial biological applications.

Microfluidic systems of multiple configurations may be used as describedbelow. Importantly, there is a wide range of microfluidics that can beused in conjunction with this system as a whole(QCLs+microfluidics+mid-IR detectors to measure individual cellproperties). Most of these must be modified to be applicable to thepresent invention through the use of substrates (such as top and bottomcaps or wafers that confine the fluid in the microfluidic structures)that transmit mid-IR light generated by the QCLs.

Multiple embodiments of QCL(s) in the system and mid-IR detectors arepossible, and may depend on the application. In an example, one or morebroadly-tunable QCLs may be used to cover a wide mid-IR spectrumcorresponding to multiple molecular fingerprints for researchapplications. Beams may be combined by use of half-mirrors (withaccompanying loss), thin film interference filters or diffractiongratings. Such a configuration may be used to gather a complete spectralsignature for a cell, for instance, where a new QCL-based measurement isbeing developed. Once useful spectral features may have been identified,such a system based on tunable QCLs may be set up with each tunable QCLtuned to a peak, and then cells may be interrogated at higher speed,such as speed that may be used in a clinical system.

Narrowly tunable lasers may be used to interrogate a specific spectralfeature, where derivative with wavelength information is important. Forinstance, rapid scanning over a small range corresponding to peakabsorption of a cell constituent may significantly improve accuracy insome cases where absolute absorption is highly variable due to otherfactors.

Lower-cost, fixed QCL lasers such as distributed feedback, or DFB-QCLsmay be used in systems where the sought-after spectral features are wellknown. In the simplest configuration, a single QCL and detector may beused to measure a chemical concentration within a cell. In the casewhere multiple constituents absorb at the signal wavelength, additionalfixed QCLs may be added to the system to make reference measurements and“back out” the effect of those non-target constituents. For example, RNAand DNA share several absorption peaks, and to make an RNA concentrationmeasurements in a cell, it is most likely necessary to measure twowavelengths and look at the relative absorption levels rather thanmeasure a single absorption peak corresponding to RNA.

A number of configurations may be possible for the mid-IR detectorscorresponding to the QCLs. In other embodiments, a plurality of detectortypes may be used, including mercury cadmium telluride (MCT)photovoltaic detectors, which may be liquid nitrogen cooled,thermoelectric cooler (TEC) cooled, or room temperature. Pyroelectricand other thermal detectors may also be used where cost is an issue. Oneor more detectors may be used in the system. For example, a system withtwo QCLs may use a single detector by using modulation on the QCLs whichare often used in pulsed mode. The signals corresponding to the twoQCLs, combined with the absorption of the sample, may be then measuredby the detector, and may be separated electronically based on themodulation patterns of the QCLs. Alternatively, thin film interferencefilters may be used to separate the two wavelengths and send them toindividual detectors. In any case, the detectors may have passbandfilters mounted in front of them to reject out-of-band mid-IR lightblackbody radiation from the system components, or broadband signalsrelating to the heating or cooling of the cell as it passes through theQCL and possibly visible/N ear Infra Red (NIR) beams.

In another embodiment, multiple QCLs may use completely distinct opticalpaths, either passing through the same sample volume at differentangles, or using altogether separate measurement volumes where a cellpasses through them sequentially. This simplifies the multiplexing ofwavelengths from QCLs, but does not ensure the same measurement is beingmade.

Another detection method that may be used in an embodiment of thepresent invention is photoacoustic detection. Photoacoustic measurementsmay be used with mid-IR including QCL measurements of gasconcentrations, even at very small concentrations. In this method, amid-IR pulse may illuminate a sample. Owing to absorption by specificchemical species, there is some local heating and expansion. Thisexpansion results in a shockwave which may be picked up by acousticsensors. Because the present invention uses closed liquid channels,there is the potential to use “microphones” to pick up absorptionsignals as a cell is interrogated with one or more QCLs. Suchmicrophones may be integrated into one of the wafers forming the top orbottom “cap” of the microfluidic device, or may be attached externallyto the microfluidic structure.

Another potential detection method that may be implemented inembodiments of the present invention is one that may involve measurementof passive (blackbody) emissions from the cell components, as they arestimulated (heated) using a QCL. For example, the cell may beilluminated with one wavelength corresponding to the absorption peaks ofseveral molecules, one of which is of interest, therefore a simpleabsorption measurement would not provide an accurate answer for themolecule of interest. Mid-IR radiation from the cell is then collected,and filtered using a narrow passband mid-IR filter around anotherabsorption (emission) peak for the molecule of interest (or anothermolecule/bond vibration that is tightly coupled, but not directlyaddressed by the input QCL wavelength). The vibrations induced by theprobe QCL wavelength may translate to an increase intemperature/vibrations, and these may be observed at this secondarymid-IR wavelength. This signal will of course be quite small, but may beelectronically filtered with well-known lock-in techniques.

A number of architectures are presented which may be used in the presentinvention to minimize the effect of water absorption and provideaccurate measurements of cellular biochemical components. This is not anexhaustive list, and the present invention will be applicable to otherarchitecture as well. In an embodiment, the present invention consistsof a flow cytometer in which one or more quantum cascade lasers may beused to measure vibrational absorption characteristics of single cellsin the mid-infrared wavelength range.

Living cells may be interrogated at high speed using preparations suchas traditional flow cytometer-type instruments retrofitted with QCL andmid-infrared transparent liquid handling microfluidics components builtfor cell classification and sorting that have been built withmid-infrared transparent components, substrates such as sheets, tapes ordiscs where live cells are distributed over a surface in a thin film andinterrogated by scanning, and the like. In an embodiment, the presentinvention may be used in conjunction with certain chemical or otheroperations in advance of the measurement that accentuate differencesbetween the cells to be sorted. For example, cells may be stimulatedwith temperature, light, fuel, or other stimulant to enhance biochemicalconcentrations that differentiate cells, for example, by differentiallychanging cell metabolism and therefore input or output products.

In an embodiment, the present invention may enable high-speed spermsorting in a label-free manner, and without exposing cells tohigh-energy UV photons. For example, the separation of sperm by X- andY-type is possible using the fact that the X chromosome hassignificantly more DNA content than the Y-chromosome, thereby enlargingthe overall DNA mass of the sperm cell by 2% or more in mammalianspecies. At least one QCL may be used to probe at least one absorptionband specific to DNA. In other embodiments, a plurality of QCLwavelengths may be used to measure other potentially interferingconstituents. The measurements may be made from more than oneorientation to normalize for the asymmetric shape of the sperm cellhead. In various other embodiments, the measurement process may besupplemented using other lasers in the visible or near-infrared bands tomeasure cell orientation, size, position, density and the like, as wellas additional mid-IR based lasers, such as alternately tuned QCL's. Theamount of mid-infrared light transmitted in the DNA band, as normalizedby the other measurements, may be used to compute total mass of DNA inthe sperm cell. As the amount of DNA for X- or Y-bearing cells for agiven species is very consistent, sperm cells may be sorted into X- andY-bearing populations with one or more mechanisms known in the art suchas flow cytometry-type equipment, microfluidics, methods where cells arespread onto larger substrates, cell cuvettes, and the like.

In an embodiment, the present invention may be a system consisting offluidics for delivering live cells one by one to a measurement volume,laser sources which generate beam(s) that may preferentially act on DNAmolecules within the cells, based on specific molecular vibrationfrequencies, detectors which measure the interaction of the sources withthe DNA as the cell passes the measurement volume and signal processingequipment to capture these signals and process them in order to generatean estimate for DNA quantity in a single cell.

In an embodiment, the system may include an interface that displays ahistogram of cell count vs. DNA quantity, a common plot used to analyzea sample of cells.

In an embodiment, the system may include other measurements made on theindividual cells that may further characterize the cell. These mayinclude, but are not limited to electrical impedance measurements in thefluid channel which may allow the system to estimate size orcross-section of the cell, and detect cell agglomerates which mayproduce false DNA readings, optical measurements in the visible or nearinfrared range of scattering or shape which may help determine celltype, and also measure agglomerates or vibrational optical measurementswhich may serve to quantify other biochemical constituents of the cellunder inspection, including but not limited to measuring protein orlipid concentrations to determine the rough size and type of the cell,and detect cell agglomerates.

In an embodiment, the system may include a method of sorting the cellsindividually based on the DNA content calculated, as well as anyreference signals including those described above. This sorting may bedone in one of a number of ways well known to those skilled in the artof cytometry, fluorescence-activated cell sorting, and microfluidics,including but not limited to electrostatic diversion of droplets, fluidpressure diversion of a stream in microfluidics, mechanical actuation ofmeasurement vs. output channels, and laser-based cell trapping/steering.

In an embodiment, the system may use a microfluidic chip on which atleast a source reservoir, output reservoir and measurement channel havebeen patterned. The chip may be a consumable component that is used onceper sample and then disposed or recycled. The core chip may befabricated from a material that is both biocompatible and compatibleoptically with the wavelengths used for the optical vibrational DNAmeasurement. The core chip may be mounted in a plastic carrier withlarge reservoir capacity, the plastic carrier itself may be used withmultiple core chips, where clogging occurs on the core chip and it isdesirable to dispose of these chips instead of performing an automatedunclogging procedure.

In an embodiment, the present disclosure may be used, and may bespecialized for the purpose of measuring a rate of cell division such asculture growth in a sample in accordance with the characteristic thatDNA per cell doubles in the process of division. This disclosure mayprovide for separating X- from Y-bearing sperm cells in accordance withthe characteristic that X-bearing sperm cells may carry more DNA thanY-bearing cells. This disclosure may provide for measuring occurrence ofaneuploidy in a sample of cells, such as for the purpose of detectingcancerous cells or other cells indicative of a genetic disorder. Thisdisclosure may provide for repeatedly measuring samples for the purposeof assessing effects of potential drugs on cells, such as cancer cells.This disclosure may provide for measuring the cell division ratesimultaneously with assessing drug effect. This disclosure may providefor separating potential cancer cells based on an aneuploidy measurementfrom a larger cell culture, where these cells may be rare. Thisdisclosure may provide for identifying and isolating cancer stem cellsfrom tumor tissue for the purpose of characterizing the stem cells. Suchcharacterization may enable further pharmacological study of the stemcells. This disclosure may provide for other applications where cellularDNA is a marker or potential marker for cell type, activity, orpathology.

Further, the present invention also describes a system for measuring thechemical composition/content of particles in the mid-IR using QCLs. Inan embodiment, the present system describes optical system architecturesto mitigate or harness scattering effects for the purpose of makingthese measurements. The optical system architectures minimize,compensate or harness these scattering effects with a minimum number ofwavelengths, allowing high-speed measurements.

In an embodiment, the cell sorting system described in the presentdisclosure may use a QCL that provides a wavelength which corresponds toparticular bond vibration frequencies. The QCL illuminates theparticle/cell, and if that molecule is present, the cell and/or analyteswithin the cell may absorb light at that resonant frequency. Theremaining light passing through the cell may be measured to determinethe amount of light that was absorbed and therefore the concentration ofanalyte. For example, DNA quantity may be measured by illuminating thecell with one or more wavelengths, one or more of which are at/nearresonant vibration frequencies for the DNA molecule. For sperm cells,measuring DNA may enable determining whether the sperm cell is carryingX or Y chromosomes because of the differential in DNA between X and Y(which is one of 23 chromosomes). The cell sorting system may comprisetwo or more QCLs. At least one QCL may correspond to the resonantabsorption (signal wavelength) for a target analyte. At least one otherQCL may correspond to a nearby wavelength to cancel out background noisefrom other analytes and system artifacts.

Other applications of the present invention may include, but not belimited to, high-speed cell sorting in the separation of stem cells fromother cells, including their differentiated derivatives, sorting livefrom dead cells, DNA content analysis for tumor biology, isolation ofkey cell populations in tumors, characterization of lymphoma cells,immune cell sorting, and the like.

In an embodiment, a minimally invasive cytometry system may usevibrational spectroscopy for gender selection. Sorting of sperm cellsaccording to the chromosomes they are carrying may enable a safe,accurate, label- and stain-free pre-fertilization gender selectionmethod. Certain of the cytometry systems described below enable such agender selection method. The cytometry system may include a handlingsystem that enables presentation of single sperm cells to at least onelaser source. The at least one laser source may be configured to deliverlight to the sperm cell in order to induce bond vibrations in the spermcell DNA. The at least one laser source emits at a wavelengthcorresponding to the resonant absorption for DNA and at least one lasersource emits at a wavelength used to cancel out a background signal fromother analytes and from system artifacts. The wavelength of the lightdelivered by the laser source may be greater than one third of thediameter of the sperm cell. A detection facility may detect thesignature of the bond vibrations, which may be used by an associated orintegrated processor to calculate a DNA content carried by the cell. Aprocessor architecture may use the characteristics of the transmitted orscattered light detected by the detection facility to perform acalculation or compare the characteristics of the detected light to thatproduced by known materials, such as to calculate a DNA content of acell. The calculated DNA content may be used to identify the sperm cellas carrying an X-chromosome or Y-chromosome. The processor may implementsoftware resident on an associated memory or server.

The minimally invasive cytometry system may also include a sort facilityfor sorting the sperm cell according to the identified chromosomes. Thecytometry system may further include a second light source configured todeliver light to a sperm cell within the sperm cells in order to inducea scattering signature, wherein the scattering signature is used toidentify a sperm cell characteristic and wherein the characteristiccomprises one or more of size, cell type, cell density, and cellorientation. The second light source may be one or more of a VIS, UV,and NIR laser source and measurements made by the second light sourcemay be used in gating the QCL-based vibrational measurements, in thisembodiment and others described herein. The cytometry system may furtherinclude a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively destroys or immobilizes spermcells based on the chromosome they are carrying. In this embodiment andothers described herein, the second light source can be used tosupplement the calculation of the content of the cell that is doneprimarily by the QCL signal (a second signal that can be used in ascatter plot).

In an embodiment, a cytometry method may include presenting a singlesperm cell, using a handling system, to at least one laser source, theat least one laser source configured to deliver light to the sperm cellin order to induce bond vibrations in the sperm cell DNA, and detectingthe signature of the bond vibrations, wherein the bond vibrationsignature is used to calculate a DNA content carried by the sperm cell,wherein the calculated DNA content is used to identify the sperm cell ascarrying an X-chromosome or Y-chromosome.

In another embodiment of a minimally invasive cytometry system forgender selection, the cytometry system may include a handling systemthat presents single sperm cells to at least one laser source, the atleast one laser source configured to deliver light to the single spermcell in order to induce vibrational absorption by DNA molecules of thesperm cell. A detection facility can then detect the transmitted mid-IRwavelength light, wherein the transmitted mid-IR wavelength light isused to calculate a DNA content carried by the sperm cell. Thecalculation can be done using an associated or integrated processor. Inthis embodiment as well as others described herein, the handling systemcan be a manifold/2D array. In this embodiment as well as othersdescribed herein, the handling system may include a carrier substrateupon which the cells are disposed and the carrier and/or the lasersource/detection facility translate with respect to one another. In thisembodiment as well as others described herein, the handling system maybe a microfluidic flow architecture. The microfluidic flow architecturemay include multiple microfluidic channels such that multiple singlecell flows may be measured simultaneously by the same light source(s).The calculated DNA content may be used to identify the sperm cell ascarrying X-chromosomes or Y-chromosomes. The calculated DNA content canbe used to identify an aneuploidy characteristic, such as an extra ormissing chromosome or a low DNA count. The cytometry system can furtherinclude a second light source configured to deliver light to the spermcell in order to induce a scattering signature, wherein the scatteringsignature is used to identify a sperm cell characteristic, and whereinthe characteristic comprises one or more of size, cell type, celldensity, and cell orientation. The second light source may be one ormore of a VIS, UV, and NIR laser source and measurements made by thesecond light source may be used in gating the laser based vibrationalmeasurements. The at least one laser source emits at a wavelengthcorresponding to the resonant absorption for DNA and at least one lasersource emits at a wavelength used to cancel out a background signal fromother analytes and from system artifacts. In this embodiment as well asothers described herein, the cytometry system may further include a sortfacility for sorting the sperm cell according to the identifiedchromosomes. In this embodiment as well as others described herein, whenthe system comprises more than one laser source, the laser sources maybe pulsed such that they result in discrete measurements on thedetector, such as alternately pulsed. Indeed, in any of the embodimentsdescribed herein, the at least one laser source may be pulsed. In thisembodiment as well as others described herein, a facility forelectronically separating the transmitted light by wavelength may beincluded and/or a facility for optically separating the transmittedlight by wavelength, such as with a dichroic filter and/or a grating. Inany of the embodiments described herein, multiple detectors may be usedto detect each wavelength. The cytometry system may further include acell destruction or immobilization facility, such as a laser emitting at1.5 microns, that selectively terminates or destroys sperm cells basedon the chromosome they are carrying.

In an embodiment of a system using microfluidic architecture for genderselection, a microfluidic architecture may include a fluid handlingsystem that enables a flow of sperm cells past at least one lasersource, wherein the fluid handling system comprises a facility to enablea single cell flow in a measurement volume of the microfluidicarchitecture. The at least one laser source may be configured to deliverlight to a sperm cell in the measurement volume of the fluid handlingsystem in order to induce resonant absorption by DNA at one or moremid-IR wavelengths. A detection facility detects the transmitted mid-IRwavelength light, wherein the transmitted mid-IR wavelength light isused to calculate a DNA content of the sperm cells that identifies thesperm cell as carrying X-chromosomes or Y-chromosomes. At least onelaser source emits at a wavelength corresponding to the resonantabsorption for a target analyte and at least one laser source emits at awavelength used to cancel out a background signal from other analytesand from system artifacts. The architecture may further include a sortfacility for sorting the sperm cell according to the identifiedchromosomes. The architecture may further include a second light sourceconfigured to deliver light to a sperm cell within the sperm cells inorder to induce a scattering signature, wherein the scattering signatureis used to identify a sperm cell characteristic, wherein thecharacteristic comprises one or more of size, cell type, cell density,and cell orientation. The second light source may be one or more of aVIS, UV, and NIR laser source. Gating the laser based vibrationalmeasurements may be based on the second light source measurement. Thearchitecture may further include a cell destruction or immobilizationfacility, such as a laser emitting at 1.5 microns, that selectivelyterminates or immobilizes sperm cells based on the chromosomes they arecarrying.

In an embodiment, a cytometry method may include flowing cells past atleast one laser source using a fluid handling system, wherein the fluidhandling system comprises a facility to enable a single cell flow in ameasurement volume, delivering laser light to the single cell in themeasurement volume in order to induce resonant mid-infrared absorptionby one or more analytes of the cell, and detecting, using a mid-infrareddetection facility, the transmitted mid-infrared wavelength light,wherein the transmitted mid-infrared wavelength light is used toidentify a cell characteristic.

In an embodiment, a label- and stain-free cytometry system forpre-fertilization gender selection may include a handling system thatpresents a single unlabeled and unstained sperm cell to at least onelaser source, the at least one laser source configured to deliver lightto the sperm cell in order to induce a resonant absorption by DNA withinthe sperm cell at one or mid-IR wavelengths. In this case, thewavelength of the light delivered by the laser source is greater thanone third of the diameter of the sperm cell. A detection facility maydetect the transmitted mid-IR wavelength light, wherein the transmittedmid-IR wavelength light is used to identify the sperm cell as carryingX-chromosomes or Y-chromosomes. The system may further include a sortfacility for sorting the sperm cell according to the identifiedchromosomes. At least one laser source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Thesystem may further include a second light source configured to deliverlight to the sperm cell in order to induce a scattering signature,wherein the scattering signature is used to identify a sperm cellcharacteristic, wherein the characteristic comprises one or more ofsize, cell type, cell density, and cell orientation. The second lightsource may be one or more of a VIS, UV, and NIR laser source. The systemmay further include a cell destruction or immobilization facility, suchas a laser emitting at 1.5 microns, that selectively terminates orimmobilizes sperm cells based on the chromosomes they are carrying.

In embodiments, cytometry systems for pre-fertilization gender selectionmay use substantially orientation-independent spectroscopy.

In an embodiment, a high yield cytometry system for pre-fertilizationgender selection using mid-IR spectroscopy may include a handling systemthat presents a single sperm cell to at least one laser source, the atleast one laser source configured to deliver light to the sperm cell inorder to induce resonant absorption by the sperm cell DNA at one or moremid-IR wavelengths. A detection facility may detect the transmittedmid-infrared wavelength light, wherein the transmitted mid-infraredwavelength light is used to identify the sperm cell as carryingX-chromosomes or Y-chromosomes. A sort facility for sorting the spermcell according to the identified chromosomes may achieve purities on theorder of at least 75%, greater than 90%, or at least 99%. For example,the purity of Y-chromosome carrying sperm cells may be at least 75%. Inanother example, the purity of X-chromosome carrying sperm cells may beat least 90%. At least one laser source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Asecond light source may be configured to deliver light to the sperm cellin order to induce a scattering signature, wherein the scatteringsignature is used to identify a sperm cell characteristic, wherein thecharacteristic comprises one or more of size, cell type, cell density,and cell orientation. The second light source may be one or more of aVIS, UV, and NIR laser source. Gating the laser based vibrationalmeasurements may be based on the second light source measurement. Thesystem may further include a cell destruction facility, such as a laseremitting at 1.5 microns, that selectively terminates sperm cells basedon the chromosomes they are carrying.

In another embodiment, a low energy cytometry system forpre-fertilization gender selection with diminished risk of cell damagemay include a handling system that presents a single sperm cell to atleast one laser source, the at least one laser source configured todeliver photons with an energy of less than 1 eV to the sperm cell inorder to induce a bond vibration in DNA of the sperm cell. A detectionfacility may detect the transmitted photon energy, wherein thetransmitted photon energy is used to identify the sperm cell as carryingX-chromosomes or Y-chromosomes. The system may further include a sortfacility for sorting the sperm cell according to the identifiedchromosomes. At least one laser source emits at a wavelengthcorresponding to the resonant absorption for DNA and at least one lasersource emits at a wavelength used to cancel out a background signal fromother analytes and from system artifacts.

Cytometry systems also described herein enable single cell study andinspection. Such systems may include light sources that induce resonantabsorption in the mid-IR wavelength region, such as a QCL laser.However, these systems may also include other light sources and othertechnologies, such as fluorescence-activated spectroscopy systems andmicrofluidic architectures.

In a further embodiment, a minimally invasive cytometry system withlaser inspection of single cells for cancer detection may include ahandling system that presents a single cell to at least one lasersource, the at least one laser source configured to deliver light to thecell in order to induce vibrational bond absorption in one or moreanalytes within the cell and a detection facility that detects themid-infrared wavelength light transmitted by the cell and identifies thecell as either cancerous or non-cancerous. The system may furtherinclude a sort facility for sorting the cell according to its status. Atleast one laser source emits at a wavelength corresponding to theresonant absorption for a target analyte and at least one laser sourceemits at a wavelength used to cancel out a background signal from otheranalytes and from system artifacts. The system may further include asecond light source configured to deliver light to the cell in order toinduce a scattering signature, wherein the scattering signature is usedto identify a cell characteristic, wherein the characteristic comprisesone or more of size, cell type, cell density, and cell orientation. Thesecond light source may be one or more of a VIS, UV, and NIR lasersource and measurement with the second light source may be used ingating the laser based vibrational measurements. The system may furtherinclude a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively terminates or immobilizescells based on the identification.

In an embodiment, a cytometry system with a laser source, acousticdetection facility, and micro-fluidic cell handling system may beconfigured for inspection of individual cells. The cytometry system mayinclude a micro-fluidic cell handling system that enables a flow ofcells past at least one laser source, wherein the handling systemcomprises a facility to enable a single cell flow in a measurementvolume of the microfluidic architecture, the at least one laser sourceconfigured to deliver light to a single cell in the measurement volumein order to induce resonant mid-IR vibrational absorption by one or moreanalytes, leading to local heating that results in thermal expansion andan associated shockwave. An acoustic detection facility detects theshockwave emitted by the single cell. The magnitude of the shockwave isindicative of a cell characteristic. The characteristic may be aquantity of a nucleic acid, a protein, a lipid, a nutrient, and ametabolic product. The micro-fluidic cell handling system furthercomprises a filter that excludes cells based on at least one of a shape,a size, and a membrane integrity. The cytometry system may furtherinclude a sort facility for sorting the single cell according to theidentified characteristic. At least one laser source emits at awavelength corresponding to the resonant absorption for a target analyteand at least one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts.

In an embodiment, a minimally invasive inspection system may use mid-IRvibrational spectroscopy for high throughput, high accuracy cytometry.The system may include a handling system that enables high throughputpresentation of single live cells to at least one light source, the atleast one light source configured to deliver light to the live cell inorder to induce vibrational bond absorption in one or more analyteswithin the cell at one or more mid-IR wavelengths. A mid-infrareddetection facility may detect the transmitted mid-infrared wavelengthlight, wherein the transmitted mid-infrared wavelength light is used todetermine a cell characteristic comprising one or more of chemicalcomposition, size, shape, and density by comparing the detected resultsto that of known cells or materials under similar conditions. Thethroughput of live cells may be at least 1 cell per second, at least 10cells/sec, at least 100 cells/sec, at least 1,000 cells/sec, at least4,000 cells/sec or at least 10,000 cells/sec. In this embodiment and inother embodiments described herein, at least one light source emits at awavelength corresponding to the resonant absorption for a target analyteand at least one light source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. In thisembodiment and in other embodiments described herein, the system mayfurther include a second light source configured to deliver light to thecell in order to induce a scattering signature, wherein the scatteringsignature is used to identify a cell characteristic, wherein thecharacteristic comprises one or more of size, cell type, cell density,and cell orientation. The second light source may be one or more of aVIS, UV, and NIR laser source. The wavelength of the light delivered bythe light source may be greater than one third of the diameter of thecell. In this embodiment and in other embodiments described herein, thesystem may further include a cell destruction facility, such as a laseremitting at 1.5 microns, that is used to selectively terminate cellsbased on the characteristic.

In an embodiment, a low energy cytometry system with diminished risk ofcell damage may include a handling system that presents a cell to atleast one laser source, the at least one laser source configured todeliver photons with an energy of less than 1 eV to the cell in order toinduce a bond vibration in DNA of the cell. A detection facility maydetect the transmitted photon energy, wherein the transmitted photonenergy is used to identify a DNA characteristic of the cell.

In an embodiment, a minimally invasive cytometry system using QCLvibrational spectroscopy for differentiation of pluripotent stem cellsfrom functionally differentiated cells based on inspection of singlecells may include a handling system that presents a single cell to atleast one laser source, the at least one laser source configured todeliver light to the single cell in order to induce vibrational bondabsorption in one or more analytes within the cell. A detection facilitymay detect the transmitted mid-infrared wavelength light, wherein thetransmitted mid-infrared wavelength light is used to identify thedifferentiation status of the cell as either pluripotent or functionallydifferentiated. The system may further include a sort facility forsorting the cell according to its differentiation status. At least onelaser source may emit at a wavelength corresponding to the resonantabsorption for a target analyte and at least one laser source emits at awavelength used to cancel out a background signal from other analytesand from system artifacts. The system may further include a second lightsource configured to deliver light to the cell in order to induce ascattering signature, wherein the scattering signature is used toidentify a cell characteristic, wherein the characteristic comprises oneor more of size, cell type, cell density, and cell orientation. Thesecond light source may be one or more of a VIS, UV, and NIR lasersource. The system may further include gating the laser basedvibrational measurements based on the second light source measurement.The system may further include a cell destruction or immobilizationfacility, such as a laser emitting at 1.5 microns, that is used toselectively terminate or immobilize cells based on the differentiationstatus.

In an embodiment, a cytometry system with a mid-infrared laser source,mid-infrared detector, and micro-fluidic cell handling system configuredfor inspection of individual cells may include a fluid handling systemthat enables a flow of cells past at least one mid-infrared lasersource, wherein the fluid handling system comprises a facility to enablea single cell flow in a measurement volume. The at least one lasersource may be configured to deliver light to the single cell in themeasurement volume in order to induce resonant mid-infrared absorptionby one or more analytes of the cell. A mid-infrared detection facilitymay detect the transmitted mid-infrared wavelength light, wherein thetransmitted mid-infrared wavelength light is used to identify a cellcharacteristic. The characteristic may be a quantity of at least one ofa nucleic acid, a protein, a lipid, a metabolic product, a dissolvedgas, and a nutrient. The fluid handling system may comprise amicrofluidic architecture. At least one laser source emits at awavelength corresponding to a resonant absorption for a target analyteand at least one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Thesystem may further include a sort facility for sorting the single cellaccording to the identified characteristic. The fluid handling systemmay further include a filter that excludes cells based on at least oneof: a shape, a size, and membrane integrity. The system can furtherinclude a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively terminates or immobilizescells based on the identified characteristic. A second light source maybe configured to deliver light to the cell in order to induce ascattering signature, wherein the scattering signature is used toidentify a cell characteristic, wherein the characteristic comprises oneor more of size, cell type, cell density, and cell orientation. Thesecond light source may be one or more of a VIS, UV, and NIR lasersource. The system may further include gating the laser basedvibrational measurements based on the second light source measurement.

In an embodiment, a mid-IR spectroscopy cytometry system with selectivecell destruction capability may include a handling system that presentsa live cell to at least one laser source, the at least one laser sourceconfigured to deliver light to the live cell in order to induce resonantmid-infrared absorption by at least one analyte within the cell. Amid-infrared detection facility may detect the transmitted mid-infraredwavelength light, wherein the transmitted mid-infrared wavelength lightis used to determine a cell characteristic by analysis. Where analysiscan include comparing detected results to results for known cellcharacteristics. A cell destruction facility may selectively terminatecells based on the characteristic. The cell characteristic may includeone or more of a nucleic acid quantity, a nucleic acid type, a chemicalcomposition, a size, a shape, and a density. The cell destructionfacility may be a laser emitting at 1.5 microns. At least one lasersource emits at a wavelength corresponding to the resonant absorptionfor a target analyte and at least one laser source emits at a wavelengthused to cancel out a background signal from other analytes and fromsystem artifacts. The system may further include a second light sourceconfigured to deliver light to the live cell in order to induce ascattering signature, wherein the scattering signature is used toidentify a live cell characteristic, wherein the characteristiccomprises one or more of size, cell type, cell density, and cellorientation. The second light source may be one or more of a VIS, UV,and NIR laser source. The wavelength of the light delivered by the lasersource may be greater than one third of the diameter of the sperm cell.

In an embodiment, a mid-IR spectroscopy system may include lasertweezers for moving cells into position for measurement with a lightsource that induces resonant absorption in an analyte within the cell.

In an embodiment, a mid-IR spectroscopy system with fluidic featuresthat pre-filter cells to obtain cells of appropriate size may include afluid handling system that enables a flow of cells past at least onelaser source, wherein the fluid handling system comprises a filter thatexcludes cells from a measurement volume of the fluid handling systembased on size and/or shape, the at least one laser source configured todeliver light to a single cell in the measurement volume in order toinduce resonant absorption in at least one analyte within the cell. Amid-infrared detection facility may detect the transmitted mid-infraredwavelength light. At least one laser source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Thesystem may further include a second light source configured to deliverlight to the cell in order to induce a scattering signature, wherein thescattering signature is used to identify a cell characteristic, whereinthe characteristic comprises one or more of size, cell type, celldensity, and cell orientation. The second light source may be one ormore of a VIS, UV, and NIR laser source.

The wavelength of the light delivered by the laser source may be greaterthan one third of the diameter of the sperm cell. The system may furtherinclude a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively terminates or immobilizescells based on the transmitted mid-infrared wavelength light.

In an embodiment, a cellular DNA measurement system may include ahandling system that presents a single cell to at least one lasersource, the at least one laser source configured to deliver light to thecell in a measurement volume of the handling system in order to induceresonant absorption in the DNA within the cell. A mid-infrared detectionfacility may detect transmitted mid-infrared wavelength light, whereinthe transmitted mid-infrared wavelength light is used to calculate thecellular DNA content. The DNA content may be used to identify cell cyclestatus in a plurality of cells and the cell cycle status for a pluralityof cells is used to determine a growth rate of the cells. The DNAcontent may be used to determine aneuploidy, wherein the aneuploidycharacteristic is identified by a low DNA count, an extra chromosome,and/or a missing chromosome. The system may further include a sortfacility for sorting the cells according to the aneuploidycharacteristic. The handling system may further include a filter thatexcludes cells based on a characteristic. At least one laser sourceemits at a wavelength corresponding to the resonant absorption for atarget analyte and at least one laser source emits at a wavelength usedto cancel out a background signal from other analytes and from systemartifacts.

In an embodiment, a cellular DNA measurement system may include ahandling system that presents a fluorescently labeled single cell to oneor more of a visible or UV laser source and a handling system thatpresents the fluorescently labeled single cell to at least onemid-infrared laser source. The at least one mid-infrared laser sourcemay be configured to deliver light to the cell in order to induceresonant absorption in the DNA within the cell. A mid-infrared detectionfacility may detect transmitted mid-infrared wavelength light, whereinthe transmitted mid-infrared wavelength light is used to calculatecellular DNA content. A visible light detection facility may detect thefluorescing label.

Mid-IR based systems described herein enable single particle study andinspection. Such systems may include light sources that induce resonantabsorption in the mid-IR wavelength region, such as a mid-infrared laseror a QCL laser. However, these systems may also include other lightsources and other technologies, such as fluorescence-activatedspectroscopy systems, microfluidic architectures, additional optics,scattering analysis, and the like.

In an embodiment, a single particle QCL-based mid-IR spectroscopy systemwith differential numerical aperture optics for emitted and scatteredlight may include a handling system that presents a single particle toat least one quantum cascade laser (QCL) source, the at least one QCLlaser source configured to deliver light to the single particle in orderto induce resonant mid-infrared absorption in one of the particle or atleast one analyte within the particle. The system may also include anoptic to capture mid-IR wavelength light transmitted through the cell,wherein the optic has a smaller numerical aperture than a focusing opticfocusing the QCL laser emission on the cell. A mid-infrared detectionfacility may detect the transmitted mid-IR wavelength light andscattered mid-IR wavelength light. The particle may be a cell. Thehandling system may further include a filter that excludes particlesbased on at least one of a shape, a size, and a membrane integrity. Thesystem may further include a sort facility for sorting the singleparticle according to one of the transmitted light and scattered light.At least one QCL laser source emits at a wavelength corresponding to theresonant absorption for a target analyte and at least one QCL lasersource emits at a wavelength used to cancel out a background signal fromother analytes and from system artifacts.

In an embodiment, a single particle QCL-based mid-IR spectroscopy systemusing resonant scattering for measurement may include a handling systemthat presents a single particle to at least one quantum cascade laser(QCL) source, the at least one QCL laser source configured to deliverlight to the single particle in order to induce resonant opticalscattering based on wavelength-specific refractive index shiftsresulting from resonant bond vibration of one or more target analytes. Amid-infrared detection facility may detect the mid-infrared wavelengthlight scattered by the single particle. The particle may be a cell. Thehandling system may further include a filter that excludes particlesbased on at least one of a shape, a size, and a membrane integrity. Thesystem may further include a sort facility for sorting the singleparticle according to the scattered light. At least one QCL laser sourceemits at a wavelength corresponding to the resonant absorption for atarget analyte and at least one QCL laser source emits at a wavelengthused to cancel out a background signal from other analytes and fromsystem artifacts. The mid-infrared detection facility may include aplurality of mid-infrared detectors deployed at multiple angles thatdetect the mid-infrared wavelength light scattered by the singleparticle.

In an embodiment, a single particle laser based mid-IR spectroscopysystem with in-droplet microfluidic system may include a handling systemthat suspends particles in or as a droplet within another liquid,wherein the handling system presents individual droplets to the at leastone mid-infrared laser source, the at least one mid-infrared lasersource configured to deliver light to a single droplet in themeasurement volume of the microfluidic system in order to induceresonant mid-infrared absorption in at least one analyte within thedroplet. A mid-infrared detection facility may detect the mid-infraredwavelength light transmitted by the droplet. The droplet and the anotherliquid may be immiscible. The particle may be a cell. When the particleis a cell, the mid-infrared wavelength light transmitted may be used tomeasure byproducts of cell metabolism in the fluid surrounding the cell.The particle may undergo a chemical reaction with the surrounding fluidin the droplet and the mid-infrared wavelength light transmitted may beused to measure the level of reactants or the products of this reaction.At least one laser source emits at a wavelength corresponding to theresonant absorption for a target analyte and at least one laser sourceemits at a wavelength used to cancel out a background signal from otheranalytes and from system artifacts.

In an embodiment, a single particle laser based mid-IR spectroscopysystem with analysis of scattering includes a handling system thatpresents a single particle tagged with a mid-IR active tag to at leastone mid-infrared laser source, the at least one mid-infrared lasersource configured to deliver light to the single particle in order toinduce resonant mid-infrared absorption in the particle or an analytewithin the particle. A mid-infrared detection facility may detect themid-infrared wavelength light scattered by the single particle. Awavelength and angle analysis of the scattered mid-IR wavelength lightmay be used to determine analyte-specific structural and concentrationinformation. The particle may be a cell. The mid-IR active tag may be aquantum dot. The handling system may further include a filter thatexcludes particles based on at least one of a shape, a size, and amembrane integrity. The system may further include a sort facility forsorting the single particle according to one or more of a transmittedmid-IR wavelength light and the scattered mid-IR wavelength light. Atleast one laser source emits at a wavelength corresponding to theresonant absorption for a target analyte and at least one laser sourceemits at a wavelength used to cancel out a background signal from otheranalytes and from system artifacts. In embodiments, mid-IR active tagsmay be used in a direct transmission measurement as well, such as withany of the embodiments described herein.

In an embodiment, a handling system may present a single particlelabeled with a mid-IR active label to at least one quantum cascade laser(QCL) source, the at least one QCL laser source configured to deliverlight to the single particle in order to induce resonant mid-infraredabsorption by the mid-IR active label of the particle. A mid-infrareddetection facility may detect the transmitted mid-infrared wavelengthlight.

In embodiments of the present invention, the wavelength for inspectionmay include wavelengths in the mid-IR band, wavelength specific to peakscorresponding to DNA vibrational modes, and the like. Wavelengthselection may be optimized such as to inhibit scattering, and the like.

In embodiments, optical architecture and system components for thepresent invention may include facilities associated with reducingoptoelectronic noise, confirming and/or modifying cell angle or positionin a measurement volume, controlling cell rate spacing, accounting fornuclear volume in analysis, and reducing and accounting for QCL supplynoise. Laser sources may include tunable QCLs, multiple tunable lasers,broadband lasers, scanning lasers, THz QCLs, QCL-on-a-chip,Vernier-tuned QCLs, single pulsed QCL, multiple pulsed QCLs, parametricoscillators, and the like. The architecture may also enable measurementsfrom various angles of capture and using beams from multiple angles. Aphase scrambling device may be used to reduce coherent artifacts. Asdescribed herein, differential aperture optics for input and output maybe used to focus the emitted light and then capture transmitted light ina wider area.

Systems with QCL sources may have a facility for separating wavelengthselectronically based on timing of pulses and relaying them to separatedetection facilities. Filters, such as dichroic filters and gratings,may be used to optically filter wavelengths. A prism or beam splittermay be used to separate wavelengths. The output from multiple sourcesmay be combined in one detector. An array of lasers may cross thechannel at different points—these can be combined and directed to asingle detector.

Reference detection facilities may also be located at an angle outsidethe main angle of capture to detect scattered light. Use of resonantscattering may be made to obtain shape and position information.Scattered light may serve as a gating signal or calibration for theprimary measurement, such as the laser based measurement. Use of mid-IRactive tags and reagents with laser interrogation may enable an analysisof scattering (e.g. for angle, wavelength). The architecture may enablepolarization-based measurements of particles in flow.

In embodiments, form factors for the present invention may include acassette, a chip, a 2D manifold, 2D array, microfluidic architecture,flow cuvette, flow cytometer, cell-on-tape/substrate and the like.Handling systems may include laser tweezers, micro-fluidic handlingsystem (e.g. for circulating cells, micro-fluidic flow systemcomponents, charge applied to cell for selection), fluid flowcomponents, with tracers, with additives, with anti-reflective coating,with labels (e.g. quantum dot labels), pre-sorting particles based onsize, a scanner system where the source moves relative to the detecteditem rather than moving the item, a microfluidic manifold (cells moverelative to each other) and the like. The scanner system may includeformats such as Cell on tape, Cell on substrate, Cells on a 2D carrierthat is read out by translating at least one of the (i) carrier or (ii)the source and the detector. Flow architecture may be a species of amicrofluidic manifold. Multiple channels of flow and multiple detectionpoints may be present in a single system.

Vibrational spectroscopy may include direct, indirect, Raman, coherent,anti-Stokes Raman, and the like. Functional benefits may include aminimally invasive system, with no labels, with no dyes, using no UVlaser, with increased accuracy, increased throughput (e.g. orientationindependent), low coefficient of variation, and the like. Two systemsmay be put end-to-end to sort for one thing in a first system, then takethe output and do a task, such as a measurement or a second sort.

In embodiments, the present disclosure provides facilities associatedwith scattering, such as measuring scattering, mitigating the effects ofscattering, correcting for scattering, utilizing resonant Miescattering, and the like. The present invention may provide for celldestruction through the use of a laser.

In embodiments, the present invention may be applied to a variety ofapplications, including gender selection (e.g. sperm DNA assessment,such as with humans, horses, cattle, pets), motility detection (e.g.sorting for motility, integrated onto the chip), cancer detection, stemcell study and manipulation (e.g. for monitoring differentiation ofpluripotent cells into functionally differentiated cells, in harvestingstem cells, in the purification of stem cells before transfer topatients), aneuploidy detection, in the measuring of cell growth rate ina sample, measuring RNA characteristics, measuring sugarscharacteristics, measuring protein characteristics, as a DNA statisticstool, in the measurement of other particles in liquids, in reactionmonitoring, in measuring circulating tumor cells, for embryo scoring, indetermining blood count, in semen analysis, in gas monitoring, insolid-in-liquid measurement, in blood diagnostics (e.g. for malaria,parasites), in food and water contamination analysis (e.g. measuring theIR footprint for E. Coli, in emulsions (e.g. using a in-dropletmicro-fluidic system), and the like.

This disclosure provides a cytometry platform for measuringcharacteristics of particles in a flow using a mid-IR based measurementsystem and vibrational spectroscopy. The mid-IR based measurement systemmay be based on a quantum cascade laser (QCL) source delivering mid-IRwavelengths in the range of 3 to 15 microns. The QCL laser has a narrowwavelength emission comprising an easily tailored center wavelength anda low etendue, enabling measurement of a small area and narrow angle andmeasurements on the order of microseconds. For example, the power of theQCL may be ≦10 mW and a conduction band offset of about 0.1 eV. QCLvariations include multiple tunable lasers, broadband, and scanning

The cytometry platform may include a microfluidic architecture. Thecytometry platform enables direct measurement of the chemical content ofthe cell using vibrational spectroscopy. Two types of vibrationalspectroscopy may be used: (a) direct absorption where the particle isexposed to a wavelength in the mid-IR fingerprint region of about 5-12microns, which corresponds to the frequency of a bond vibration and (b)Raman spectroscopy which is an indirect way of making measurements.Coherent anti-stokes Raman spectroscopy (CARS) may also be used tomeasure vibrational bond fingerprints in cells.

The cytometry platform enables a stain- and label-free process that issafe to cells and is orientation independent, high throughput, highaccuracy, and high yield. The advantages of the cytometry platforminclude the ability to directly measure an absorption line withoutlabels or dyes/stains. Another advantage is use of light that is 20-25times less energetic light than UV light that is used in conventionalsystems and thus much less likely to damage cell. Because of thewavelength being relatively long (six times longer than UV laser), thereis less scattering and the scattering is much better behaved compared toother light sources, such as visible light. Another advantage is that asopposed to traditional FTIR that requires cells to be dried, thecytometry platform can measure cells in a liquid medium where refractiveindex differential is much lower, so scattering index is lower.

In some instances of the cytometry platform, an additional scatteringmeasurement may be made specifically to see how big and dense a cell is.For example, a blood count can be done in this fashion. With mid-IRlight and in particular light whose angle can be controlled well (with avery collimated beam), chemical composition and size information may beobtained by measuring scattering off of particles.

In embodiments of the cytometry platform, a conventionalfluorescence-activated sorting (FACS) system may be used in parallel inorder to obtain a simple binary measurement for a cell then get anaccurate numerical measurement for chemical content using the mid-IRbased measurement system.

The cytometry platform enables measurement of cellular content, such asprotein bonds and nucleic acid bonds. For example, three characteristicDNA peaks, an asymmetric PO₂ ⁻ stretch (DNA) at 1236 cm⁻, a symmetricPO₂ ⁻ stretch (DNA) at 1087 cm⁻, and a C—C deoxyribose stretch (DNA) at968 cm⁻.

The cytometry platform is engineered to reduce the QCL supply noise, theRIN noise associated with emission of the QCL, shot noise associatedwith the transmitted mid-IR energy, detector noise, and pre-amp noise.For example, at bandwidths up to 10,000 cells/second, the optoelectronicsystem noise is 3.7 ppm. Other elements of the cytometry platform werealso designed to reduce system noise, such as the selection of thechannel height, the input angle, the collection angle, the flow rate,and cell spacing.

One design of the system includes 2 or more fixed wavelength QCLs whereat least one is at a “signal” wavelength (corresponding to the resonantabsorption for a target analyte) and at least one is at a “reference”wavelength (a nearby wavelength used to cancel out background from otheranalytes, and from system artifacts). The QCLs may be in a coolinghousing and may be driven by a pulsed driver or some other kind ofdriver. A grating-based QCL tuning system may be included to tune theQCLs. Before reaching the sample, the QCLs may first traverse a pelliclebeamsplitter and adjustable aperture. Transmitted mid-IR energy isdetected by a signal detector and a reference detector. A detector maymeasure scattering and compensate for it in the main measurements. Forsuch a system, the predicted collection rate is >4,000/sec and thepredicted purity is >99%. The QCLs may have carrier frequencies so thata single detector may be used and the signal is separated by modulationfrequencies.

With wide variations in nuclear volume and cell orientation, there isvariability in the absorption peak. Selecting a wavelength that is atthe top of the curve where the strongest absorption exists may not beideal as strong absorption may make measurements more variable with cellorientation.

The cytometry platform is designed to reduce cell nucleus heating.Heating, due to DNA absorption of mid-IR energy, may be reduced and mayhave a narrow distribution even with wide nuclear volume/orientationdistribution. For example, heating may be kept to under 1K.

In the cytometry platform, a laser source such as a QCL may beconfigured to emit energy to a single cell in a measurement volume ofthe platform in order to induce resonant mid-IR vibrational absorptionby one or more analytes, leading to local heating that results inthermal expansion and an associated shockwave. The cytometry platformmay include an acoustic detector that detects the shockwave transmittedby the single cell, wherein the shockwave is indicative of a cellcharacteristic.

In the cytometry platform, a cell destruction facility may be includedto selectively terminate cells based on a characteristic. In the exampleof pre-fertilization gender selection, those sperm not exhibiting thedesired chromosomal type may be targeted for motility cessation using alaser emitting at 1.5 microns which may be used to immobilize thosesperm without damaging DNA contents. In other cases complete destructionof sperm through membrane destruction or other means may be effected,through the use of a laser or other means. The cytometry platform maytake many forms. The cytometry platform may be embodied in a mid-IRcuvette for a standard flow cytometer where cell selection is based onan applied charge. The cytometry platform may be embodied in a 2Dmanifold/array for immobilizing a plurality of cells and then measuringthem individually using the present invention, potentially repeatedly.The cytometry platform may be embodied in a system with laser tweezersthat traps cells with a visible laser and moves the cells into positionfor measurement. The cytometry platform may be embodied in amicrofluidic chip with a waveguide to capture IR light that crosses thechannel. The microfluidic chip may include fluidic features thatpre-filter cells by size. The cytometry platform may be embodied in acirculating cell culture system. The height of the channel may beoptimized so that a resonant optical field is not obtained even if theAR coatings are not robust. The mid-IR wavelength used in variousembodiments herein may be optimized for low scattering.

The mid-IR based inspection and measurement system may further include afacility for a polarization-based measurement of particles in a flow,such as of DNA especially. Even with measurement of phosphate bonds, ifleft hand or right hand polarized light is emitted, because the DNA isin a helix, an improved separation of a DNA-specific signal may beobtained by using circularly polarized light.

Now that we have described particular embodiments of the presentdisclosure, we turn to describing a set of figures that will illustratethese and other embodiments.

FIG. 1 illustrates the present invention configured in a flowarchitecture 100. The flow architecture 100 may be a minimally invasivecytometry system, a microfluidic architecture, a cell inspection system,a label free cytometry system, a dye free cytometry system, a high yieldcytometry system, a low energy cytometry system, an aneuploidymeasurement system, a growth rate measurement system, an in-dropletmicrofluidic system, and the like. The flow architecture 100 comprises alaser source 102, a detector 104, a pre-filter 108, a single cell flow110, a sort facility 112, one or more sort destinations 114, and ahandling system 118. The laser source 102 may be a QCL, a QCL array, aQCL array of multiple angles, multiple QCLs with distinct carrierfrequencies, a Vernier tuned QCL, a dual QCL/UV array, a QCL with phasescrambling facility, a mid-IR laser, a tunable QCL, a broadband QCL, ascanning QCL, a THz QCL, or any combination thereof. The detector 104,also known as a detection facility, may be one or more of a mid-IRdetector, visible/NIR scattered light detector, fluorescence detector,quantum dot label detector, detector with differential numericalaperture, reference detector for calibration, photo-acoustic detector,or a combination thereof, and may be selected in accordance with thelaser or light source. The handling system 118 may be a microfluidichandling system, a chip/cassette with or without anti-reflectivecoating, a 2D manifold/array, a laser tweezers, and the like. Thehandling system 118 may comprise a facility to enable single particle orcell presentation to the one or more laser sources 102.

In an embodiment, handling system 118 may be a fluid handling system ina microfluidic architecture. Various cytometric analyses,characterizations, measurements, diagnoses and identification may bepossible using the present disclosure such as pre-fertilization genderselection, cancer detection, reproductive cell/embryo viabilitydetection, high throughput live cell studies (optionally in combinationwith FACS), stem cell studies, selective cell destruction, measurementof aneuploidy, measurement of growth rate, measurement of DNA, RNA,proteins, sugars, lipids, nutrients, metabolic products, etc, gasmonitoring, determining food/water contamination, and the like. A samplefluid may be encased in a sheath fluid that may allow droplets to becharged. The fluid stream may be streamed out of a nozzle. The presentdisclosure uses flow architecture 100 of a path length such as 50microns or less to reduce water absorption of infrared signal. In anembodiment, the single cell flow 110 may be passed through an opticalmeasurement zone, where infrared light emitted by laser source 102passes through the single cell flow 110 including at least one firstcell type 120 and at least one second cell type 122. The opticalinterrogation may occur either after the fluid exits the convergingnozzle, as shown in FIG. 1, or when it is still within the convergingnozzle. In the in-nozzle case, the converging nozzle may be made of atleast one infrared-transparent material such as Germanium, very pureSilicon, chalcogenide glasses, Calcium Flouride, Zinc Selenide and thelike. The beam from laser source 102 passes through the fluid stream andmay be detected on the opposite side with one or more mid-infrareddetectors 104. When a living cell is detected in the stream, theabsorption of analytes within the living cell may be measured at one ormore mid-IR wavelengths as the cell moves through the beam by studyingthe transmitted mid-IR wavelength light. The signal from detector 104may be processed to yield an estimate of certain biochemicalconstituents in the cell. The absolute or relative level of theseconstituents may be used to classify the first cell type 120 and/or thesecond cell type 122.

According to methods well known in flow cytometry, the stream isactuated in a manner, such as with a piezoelectric actuator that maycause it to break quickly into discrete droplets. These droplets may begiven an electrical charge according to the cell classificationdetermined using the laser system. In an embodiment, the system mayapply an electrical charge to desirable cells, and route all otheruncharged droplets or cells to a waste bin. However, in otherembodiments, various systems may apply multiple levels of charge tocells or droplets in order to allow sorting within sort destination 114.Once assigned a charge, the cell or droplet may be attracted/repelled bycharged plates in the sort destination 114. The negatively charged cellsor droplets are attracted by the +Ve plate, and sorted into one outputcontainer; the positively charged droplets are attracted by the −Veplate. The cells or droplets whose readings are inconclusive (where thedroplet contains no cell, or where the droplet contains more than onecell) can be sorted into a waste container.

In an embodiment, the configuration in FIG. 1 can be applied topre-fertilization sperm cell sorting for gender, for example. Spermcells would be interrogated by laser light source 102 that is tuned tothe absorption of DNA, although laser light source 102 can be tuned toother relevant cellular matter. In addition a visible laser can be usedto measure scatter from the cell. The visible laser and cellular matterlaser wavelengths can be used to determine if a single sperm cell ispresent, and possibly the orientation of the cell. The absorption of thewavelength for laser light source 102, and the associated detector 104,may be integrated and processed together with the other readings todetermine the total mass of DNA in the cell. The 2-5% differential inDNA mass between cells carrying the X- and Y-chromosomes is then used tosort cells into X-bearing and Y-bearing samples. Inconclusivemeasurements, multi-cell droplets, and droplets without cells can besent to a waste container. In other embodiments, the system may befurther simplified for this application by having only “selected cells”and “waste” outputs. In some embodiments, spectral measurements of spermcells have been used to measure the extent of any chromosomal/DNA damagein a cell. Cells that have unusual spectra in their DNA fingerprint maytherefore be discarded. Similarly, cells showing unusual ratios of othercellular matter compared to DNA matter may indicate lack of viability ordamage to the cell, and this information can be used to reject the cell.

FIG. 2 shows a potential configuration of laser source 102 tointerrogate a sample stream, in either a flow cytometer 100configuration as shown in FIG. 1, or in another configuration wherecells are presented in a fluid channel such as in a microfluidic chipsystem. In this embodiment, a visible laser 202 may be used to detectthe arrival of a cell in the stream by its scattering signature. Thescattering signal may encode other information about the cell includingsize, features that may help indicate the cell type, or orientation.

The visible scattering signal may then be used to trigger QCL 204operation where one or more pulsed QCLs are used. The advantage ofpulsed QCLs running at a low duty cycle is that they may producesignificantly higher power for the short period in which the cell is inthe measurement location, allowing for a higher signal-to-noise ratio inthe measurement.

The visible laser 202 measurement may be configured such that it detectscells ahead of the QCL 204 volume for a number of reasons as it mayallow the QCL 204 to be turned on and stabilize in terms of power andwavelength before the spectral measurement commences, it may also bedesirable that the QCL-based measurement begins before the cell arrives,and coincidentally with the visible laser, in the measurement volume, inorder to have baseline infrared measurements before and after the cellis measured. However, in other embodiments, a separate detector may beused on the output of the QCL 204 to normalize out laser power. Inalternate configurations, the “visible” and mid-infrared measurementvolumes may be the same, and beams may be combined into a single beam.The visible beam may be enlarged along the flow axis to produce a signalthat is longer than the QCL-based measurement. The visible measurementmay measure scatter to one or more detectors 206, and may even be usedto produce an image or pseudo-image of the cell in order to measure sizeor orientation. Cell orientation may be critical information for sortingbased on QCLs 204. For example, sperm cells, which pack DNA very tightlyinto their body, may be asymmetric. Transmission measurement through thelong axis of the cell may result in a significantly different absorptionmeasurement than through the short axis. With the aid of one or morevisible beams and their scattered signals, cell orientation may bedetermined to process the QCL-based measurement more accurately.

FIG. 3 illustrates a simplified example of mid-infrared spectra for aflow such as those described in FIGS. 1 and 2. The example showsinfrared transmission of three constituent materials within the flowwhich includes water, DNA and cellular components, however, there may bemany cellular components other than DNA, and DNA and each characteristictransmission spectrum may have many features.

In an embodiment, three QCL wavelengths may be used: one to measurewater absorption through the stream, one to measure cellular componentsother than DNA, and one to measure the DNA signal. The three absorptionspectra may be overlapping, in which case a 3-QCL approach is desirablein order to strip out the DNA signal. In an embodiment, the outputs ofthe 3 QCLs may be combined into a single beam that is sent through thesample, and then broken into separate wavelengths using thin filmfilters and the like, and detected by 3 separate mid-infrared detectorssuch as cooled Mercury-Cadmium-Telluride (MCT) detectors. The absorptionof the water is calculated first, to normalize the measurement of thenon-DNA cellular constituents; in the case where the cellularconstituent absorption overlaps with the DNA absorption spectrum, thissignal is then used to normalize the signal received from theDNA-specific wavelength detector. In an embodiment, the signalcorresponding to non-DNA components may be used separately to classifythe cell type, orientation, and the like. In other embodiments, abroadband QCL source may be used to produce mid-infrared light coveringall the relevant features, and a similar 3-detector configuration may beused. Another configuration may use a scanning tunable QCL that rapidlyscans the wavelength range of interest, in addition to a singledetector. Another configuration may be the use of a broadband QCL sourceplus a tunable detector system.

FIG. 4 shows an example configuration of a system 400 interrogatingcells 420 in a flow 410, which is shown in cross-section. In thisembodiment, multiple QCLs 430 and 432 provide infrared wavelengths λ₁and λ₂ may be combined with a visible wavelength from a visible laser440 to interrogate cells 420. The wavelengths may be combined by mirror470 and dichroic filters 472 and 474, and then separated and directed atdetectors 450, 460 and 462, using dichroic filters 484 and 480 andmirror 482, respectively. The QCL wavelengths λ₁ and λ₂ may be used inthis case to normalize for water, or to measure relative constituentswithin the cell 420. The visible wavelength provided by visible laser440 may be used to detect the cell 420, and in other ways describedearlier.

FIG. 5 shows another embodiment of the present invention, in which aflow 510 (shown in cross section) and a cells 520 is measured frommultiple angles. As shown in FIG. 4, the system of FIG. 5 may use one ormore QCL wavelengths and visible/near infrared wavelength(s).Measurements using one or more paths through the flow 510 may be used tocalculate or normalize for cell 520 orientation or precise positionwithin the fluid flow 510. In the embodiment of FIG. 5, a QCL 530provides mid-infrared light to a beam splitter 572 which reflects afirst portion of the light toward the flow 510, where the cell 520 isinterrogated and the light passes on to detector 560. A second portionof the light from QCL 530 passes through the beam splitter 572 and isreflected by mirrors 570 and 571 so that it is directed at the flow 510and interrogates cell 520 from a different angle, after which it isdetected by detector 560. Such multi-angle measurements may result insignificantly higher precision measurements of cellular components. Inother embodiments, visible beams where laser sources and detectors aresignificantly less expensive may be used from multiple angles toprecisely localize the cell in the flow and/or measure its orientationto normalize measurements made using the QCL source(s) and associatedmid-infrared detector(s). The embodiment shown in FIG. 5 may be extendedto multiple angles through the sample. If additional power is needed toprovide high enough (signal-to-noise) SNR, multiple QCL sources may beused. As described above, this may be supplemented with visible/nearinfrared measurements from multiple orientations, which may use the samebeam paths, or a separate set of beam paths.

FIG. 6 illustrates an example set of signals obtained from a system witha visible laser, and three different QCL lasers wherein the QCL lasersare setup to measure different aspects of the flow and cells in theflow. The first (top) signal graph is from a visible laser andassociated detector measuring scatter from a cell as it passes throughthe measurement volume, wherein the dip in signal indicates the presenceof a cell. The second signal graph is for a QCL that is used to measurewater absorption through the stream (essentially measuring the pathlength through the stream). The third signal graph is for a QCL is usedto measure general cellular components. The fourth (bottom) signal graphis for a QCL that is tuned specifically to the absorption band for DNA(for example the O-P-O stretch band at 1095 cm^-1 DNA/RNA). In themanner described above, the signals are analyzed in combination tonormalize each of the signals and thereby isolate the signal of interest(which may be the DNA content of the cell).

FIG. 7 shows another embodiment of the present disclosure, where cellsare measured in a dry state. In this embodiment, a continuous tape 707is used as a substrate for the cells 720 for measurement and separation.The tape may be metal-coated to provide high reflectivity in themid-infrared. This example may be shown for sperm cells 721, which maywithstand some level of desiccation. Sperm cells 721 may be spread ontothe tape 707 from a liquid reservoir 730—a “squeegee” type nozzle 732may be used, potentially with features that may provide the sperm cellswith a common orientation on the measurement tape. The sample may bedried rapidly but carefully with an air dryer 742, so that it does notdestroy the cells to a point where very little extraneous fluid is leftaround the cells. A sensor head 750 containing QCL lasers, detectors,and visible/near infrared lasers/detectors may be then scanned over thetape. Absorption of the QCL wavelengths may be measured to determine, ina manner described above, the absolute amount of DNA contained in thecells and potentially other information regarding the cells. Thisdetermined information can be used to identify cells to be saved 723 andcells to be rejected 722.

In a further embodiment, rejected cells 722 or regions that may berejected are then covered using an ink-jet type device 760 capable ofapplying a substance that fixes the rejected cells to the substrate overa controllable area somewhat bigger than a cell but smaller than thedistance between cells. Optionally, the tape 707 may then be rehydratedusing a rehydrator 770 to preserve the cells to be saved 723. The tape707 then runs into an output reservoir 780, where the cells to be savedcan be extracted into a liquid. The tape 707 with the rejected cells 722fixed to it goes into a waste container. The advantage of thisconfiguration may be that the resulting piece of equipment may be verysmall, and take advantage of components already developed for low-cost,high-speed scanner and printer systems. As the cost of QCL componentsmay be reduced, this makes possible the use of low-cost, compact systemsfor clinics or even home use.

In addition, the removal of liquids from the “sample stream” greatlyincreases the transmission of mid-infrared light and potentiallyimproves the signal to noise ratio (SNR) of the system. The basic systemillustrated in FIG. 7 may be configured in alternate manners in linewith the present invention. Other embodiments may include use offreezing rather than drying to fix the cells in place and prepare formeasurement; use of selective unfreezing and removal in order toaccomplish the selection process; and no use of drying or freezing butallowing a thin layer of liquid or gel to remain on the surface of thetape by hardening of the liquid/gel by any means in order to fix certaincells to the tape while others are extracted, including selectivedrying, exposure to radiation that hardens a gel, and the like. Anotherembodiment may include use of a laser or other mechanism to destroy ordisable cells that may be determined by use of the present invention tobe of a type not desired to be saved. Potential subsequent filtering toseparate dead from live cells may also be included.

FIG. 8 shows the application of the present invention embodied using amicrofluidic-type cell sorting system. Such devices have beendemonstrated for cell sorting using conventional dye plus UV-activatedfluorescence classification of cells. In an embodiment, the microfluidicdevice 800 is configured in a basic manner, with an input well 805 andtwo output wells 807 and 809. Sorting at the junction 811 isaccomplished in one of the several ways known to those in the field,including but not limited to electrical fields, lasers, magnetic means(in conjunction with magnetic beads), or fluidic pressure ports. In anembodiment, the system may be configured to allow interrogation in themeasurement volume 813 using one or more QCLs 815 from the top, andsimultaneous interrogation using a visible laser 817 from the side. Thismay be achieved, for example, by using a glass substrate as the lowerhalf of the device (transmissive in the visible) and high-purity siliconas the upper “lid” for the device (transmissive in the mid-infrared).The lids may be coated to improve optical performance; for example, theglass in the channel may be coated with a (visible light) transparentconductive layer that strongly reflects mid-infrared radiation tomaximize the mid-IR signal returned to the mid-IR detector(s). Asdescribed above, the visible laser 817 may be used to detect a cell 820in the measurement volume 813, and then trigger pulsed operation of theQCL 815. The mid-infrared light from one or more QCLs 815 passes throughthe cell, is reflected by the bottom of the microfluidic channel, andinto the mid-infrared detectors 819. The integrated signal as the cellpasses through the detection volume may be used to classify the celltype, and to control the sorting mechanism at the junction 811.

FIG. 9 shows a portion of a very basic embodiment of the presentinvention which is a microfluidic system for live cell measurements. Amicrofluidic chip with input 900 and output 902 wells may beconstructed, in manners well known in the industry, here with threelayers: a top cap 912 in which the input 900 and output 902 wells areetched, a bottom cap 910, and a patterned layer 914 in whichmicrofluidic features are patterned. This layer 914 may consist of anyof a number of materials, for example Polydimethylsiloxane (PDMS), whichis readily patternable (by imprinting, for example) and biocompatible.In addition, wells 900 or 902 may be formed in this materialcorresponding to those patterned in the top cover. One or more channels904, which may be tapered to prevent clogging, may be patterned toconnect the input 900 and output 902 wells. A portion of the channel 904constitutes the measurement volume 908 where a mid-infrared beamproduced by one or more quantum cascade lasers (QCLs) may be focused onthe channel 904 and transmitted or reflected to one or more mid-infrareddetector(s), such as a mercury cadmium telluride (MCT) photodetector.Cells passing through or positioned in the measurement volume 908 maycause absorption of specific wavelengths of mid-infrared lightcorresponding to molecular vibration modes. The absorption measured bythe system at these wavelengths may be used to chemically and thereforebiologically characterize the cells as previously described.

One or both of the caps 910 and 912 and may be a material that transmitsthe mid-infrared wavelength(s) of interest. Standard glasses used inmicrofluidics may absorb these wavelengths. To build this architecturefor use with QCLs in the molecular “fingerprint region” (2-20 micronswavelength, where molecules have their fundamental vibration modes), aninfrared-transmissive material, for example ZnSe, may be used for layers910 or 912. Other materials which may be used may include at least oneof Ge, Si, BaF₂, ZnS, CaF₂, and KCl. Certain materials such as BaF₂ orZnSe may be transparent in at least portions of the visible light range,which may be advantageous for systems where visible light guidance,observation, measurements or manipulation is used in the measurementvolume 908 as previously described. The visible light may include shortwavelength light such as ultra-violet, visible, NIR of up to 2 microns.In certain embodiments of the present invention, 1.5 microns light maybe used to ensure compatibility with the infrared-transmissivematerials.

If the system is built for transmission measurements through the channel904 in the measurement area 908, then both top and bottom caps 910 and912 must be transparent in the mid-infrared. An alternativeconfiguration can include one reflective cap and one transmissive cap,where the mid-IR light from the QCL(s) passes through one cap (912 or910), passes through the measurement volume 908, reflects off theopposite cap (910 or 912), passes through the measurement volume 908 asecond time, and then exits the cap and is collected by a mid-IRdetector (similar to the system shown in FIG. 8). In this case, only oneof the cap layers is required to transmit mid-IR light. For exampleSilicon may be used depending on the precise wavelengths being measured.High-purity Silicon such as float-zone Silicon may be desirable toreduce absorption losses in certain ranges. The opposite reflective capmay use a standard microfluidic material, such as glass. In thisarchitecture, it may be desirable to coat the glass with a mid-IRreflective layer at least in the measurement region 908. For example,even a thin layer of metal may be highly reflective in the mid-IR.Alternatively, a conductive oxide such as indium tin oxide (ITO) may beused in order to reflect mid-IR but be transparent in the visible range,in order to allow visible or near-infrared (NIR) access to themeasurement volume 908 from the opposite side. The flow of cells throughthe channel 904 and measurement area 908 may be controlled in a numberof manners well known in microfluidic systems, including pressuredifferentials between wells 900 and 902 or an electrical potentialbetween wells 900 and 902. The system may be configured to provide acontinuous throughput of cells through the measurement area 908 forapplications such as cell type counting or population statistics, or toallow precise positioning and stationary measurements of cells in thisarea for high-resolution spectral and other inspection of cells for R&Dapplications. The structure illustrated may be replicated or multiplexedusing multiple fluidic channels, which may be interrogated in parallelor sequentially by QCL-generated mid-IR light. Parallel channels mayallow for higher system throughput, and/or redundancy in case ofclogging.

In an embodiment, the present disclosure provides a method for disablingor destroying cells in the system. For example, a laser with sufficientpower to destroy key portions of the cell may be used to disable thecell. The disabled cell then flows into the output, where it may beseparated using filtering or other means, or left in the sample if itdoes not disrupt the function of the live cells. An example of such anapplication may be characterization and selection of sperm cells. Thosecells which meet the selection criteria (for gender, for instance) maybe allowed to flow through the channel unchanged, those that do not meetthe section criteria may be irradiated with a pulse of visible orinfrared light which damages their cell membrane or propulsionmechanisms. Subsequently, a “swim-up” filter which enables motile spermto be extracted may be used to collect the undamaged sperm cells.Alternatively, the entire sample may be used and only the undamagedsperm are able to fertilize the egg. A system based on the presentdisclosure would flow a suspension of sperm cells through one or moremicrofluidic channels. In the measurement volume of a channel, one ormore QCL beams would be used to determine the volume of DNA present inthe sperm cell, and thereby determine whether the sperm cell is carryingX or Y chromosomes as previously described and optionally whether thereare mid-infrared spectral indicators for other characteristics ofinterest. The mid-infrared measurement may be triggered and/orsupplemented by a low-power visible/NIR beam which may measure cellscattering or size, based on the X/Y characterization in the mid-IR andother markers in the mid-IR and/or visible/NIR. Those sperm which aredetermined to be desirable may flow through to the output withoutfurther intervention. Those that are determined to be undesirable may beilluminated within the measurement area, or immediately after it with apulse of light which immobilizes or otherwise damages it. In anembodiment, the present invention may utilize light in the near infraredor short-wave infrared range to immobilize the sperm. In an embodiment,a mechanism for separating motile from non-motile sperm may be builtdirectly into the microfluidic device in this system.

Another example where such a selective-kill mechanism may be employed isin stem cell therapies. For example, when a suspension of differentiatedcells grown from pluripotent stem cells may be prepared for deliveryinto the subject, it is important to remove or disable residual stemcells, which may grow into tumors within the target organ. In this case,a filtering system based on the present invention would inspect a flowof cells in one or more microfluidic channels, and interrogate thesecells with mid-IR beams from one or more QCLs. Based on the observedspectral characteristics, the biochemical makeup of cells passingthrough the filtering system can be determined. The cells that aredetermined to be residual stem cells and cells that are not the desiredtype of differentiated tissue are destroyed using a laser pulse as theypass through the filtering system. In this manner, cells with thedesired differentiated tissue receive minimal handling and only verylow-energy mid-IR radiation exposure, while cells which could causeabnormal growths if delivered intact to the target tissue are renderednon-functional or destroyed.

A similar system may be used if a closed-circuit system is built for theculturing, growth and filtering of stem cells or other cell types. Cellsmay be continuously run through a filtering system that inspectscellular biochemical fingerprints and terminates cells that do not meetthe application requirements. Again, applications of such systems mayinclude regenerative medicine based on stem cells. Another potentialapplication of such a system may be in high-throughput “evolutionary”processes where specific cell function is being targeted, and thisfunction or its byproducts may be observed using mid-IR spectroscopictechniques. For example, synthesis of specific chemical particles orcells may be the functional target, in this case a system based on thepresent invention may flow, with high throughput, particles or cellsthrough microfluidic channels where they are interrogated using QCL(s)and mid-IR detectors. Particles or cells showing promise can berecirculated into the system without change, those that do not showpromise can be destroyed using a laser or other potential tools such asultrasound, RF, mechanical punches, liquid jets and the like, so they donot contribute to future populations in the system.

FIG. 10a shows detail of an embodiment of a measurement volume in amicrofluidic channel 1002. Cells 1004 flow through the channel 1002 andpass a region illuminated by a mid-IR beam 1008 originating from one ormore QCLs. As the cell 1004 passes through the beam 1008, mid-IR lightis absorbed in a spectrally-dependent manner according to the molecularconstituents of the cell. In many cases, the area of the beam 1008 maybe larger than the cell 1004, and the extracted signal will correspondto an average over the cell 1004 and surrounding areas. QCLs, as opposedto traditional mid-IR sources such as hot filaments, may be able tofocus significant power into small areas, which provides a strongadvantage for this system. In many cases it may be desirable to masksurrounding areas in order to reduce the signal background. In this casea masking layer 1012 may be patterned on to one of the caps in order tocreate an aperture 1010 through which mid-IR light may pass. Thisimprovement increases contrast as the cell 1004 passes through themeasurement volume, and reduces contributions from other materials suchas the PDMS or other material used to create the fluidic channel 1002.

FIG. 10b shows the same example as 10 a but in cross-section.Specifically it shows how a mask 1012 is used to ensure only a subset1014 of the incoming mid-IR light 1008 from the QCL(s) is transmittedthrough the cell 1004, through the aperture 1010 where upon, thetransmitted light is detected by the mid-IR detectors (not shown). Inthis example, both top and bottom caps 912 and 910 must be made out of amid-IR transmissive material. If visual-range observation ormeasurements are also desired, at least one of these must betransmissive in both the mid-IR and visible ranges.

FIG. 10c shows a cross-section of an alternative embodiment that may usea reflective measurement for the mid-IR light 1008. In this example, themeasurement volume is illuminated with mid-IR light 1008 through thebottom cap 910 that is made of IR-transmissive material. A portion ofthis light passes through the measurement volume, where it may passthrough a cell 1004, and is then reflected by a patterned reflectivelayer 1018 applied to surface 1012. The reflective layer 1018 may be ametal layer, or a conductive oxide as described earlier in order toallow visible light access into the volume from the top. Reflectedmid-IR light then makes an additional pass through any cells 1004 in themeasurement volume, and then passes out through the bottom cap 910 whereit may be captured by a mid-IR detector system. The mid-IR light 1008that is not reflected passes into the top cap 912 and is absorbed. Theadvantage of this architecture may be the ability to use two differentwafer materials for the top and bottom caps 912 and 910. For example,the top cap 912 could be made of glass, which is low cost, transparentfor visible observation or measurements, and can be readily patterned toform fluid cells/ports. The bottom cap 910 may be made ofIR-transmissive material but not necessarily require visibletransmission. For example, silicon may be used for certain wavelengthranges.

FIG. 11 shows an embodiment of the invention where the microfluidics andQCL-based spectral measurement system may be combined with a moreconventional fluorescence-based measurement system. A microfluidicdevice 1100 may include channels to hold live cells 1004 that areilluminated with one or more QCLs, which may include one or more tunableQCLs 1102. The mid-IR radiation 1008 emitted by the QCL 1102 passesthrough the measurement volume and any cells 1004. The transmitted light1012 is then measured by one or more mid-IR detectors 1104. This may bedone in either transmission (as illustrated here) or reflection (asdescribed earlier) modes. Here the system may be shown to becomplemented by a laser in the UV or visible range 1108 which may emitlight 1110 that is also directed at the measurement volume and cells1004 where it excites fluorescent probes or dyes attached to specificcellular features. The resulting fluorescence 1112 may be measured usingone or more dichroic bandpass filters 1114 and associated detectors1118. Such a system configuration may allow correlation of conventionallabel-based techniques with mid-IR spectroscopic measurements. It mayalso enable combined-mode systems where labels may be used to identifyspecific cell types/features, and mid-IR spectral measurements usingQCL(s) supplement this information with spectral measurements thatenable additional information to be determined by analysis, such aspossibly for biochemical variations not possible to measure with knownlabels/dyes. Note this system may be configured, as discussed earlier,as a two-sided reflective system where IR and visible systems arepositioned on opposite surfaces of the microfluidic system and operatein reflection.

FIG. 12 shows an exemplary embodiment of the present invention where themicrofluidic subsystem includes a cell-sorting fluidic switch 1208. Aninput channel 1202 delivers cells (where the cells can be transported ina fluid) to the measurement volume 1204 where it may pass through aQCL-derived mid-IR beam from one or more QCLs, with one or morewavelengths probing the cell. The microfluidics may have beenconfigured, in manners known in the industry, to center the cells in theflow channel. Based on absorption at these mid-IR wavelengths, the cellmay be classified into one of two categories. In some embodiments, twopressure ports 1210 may be used to displace each cell to one side or theother of the microfluidic channel depending on which category the cellhas been classified into, so as to cause it to flow into one of twooutput channels 1212. Such a system may be used to accumulate one typeof cell out of a general flow of cells, or to forward one or twopopulations for further inspection and/or processing. An exampleapplication of such a configuration may include gender selection, wheresperm cells carrying X or Y DNA may be sorted into groups and one typemay be retained for fertilization. In an example of a stem cellapplication, pluripotent stem cells may be separated from differentiatedcells during extraction, or before reintroduction to a subject. Otherapplications include refinement processes where cells are cultured,possibly mutated and repeatedly measured, with certain cellsre-introduced to the culture based on their chemical “fingerprint”. Forexample, those cells which produce and therefore contain a specificproduct may be selected for, on a cell-by-cell basis. The switchingfunction in the microfluidic channel may be performed using any of anumber of techniques known to the art, including electrostatic forces,acoustic forces, optical pressure/heating of a portion of the channel,containment of cells in bubbles that may be transported within anothermedium, and the like.

FIG. 13a shows an alternative microfluidic-based embodiment of thepresent invention, where a series of microwells 1304 may be integratedinto a microfluidic flow channel/chamber 1302. This structure may be1-dimensional along a single channel or 2-dimensional along an openplane. Multiple parallel channels may be used as well. These wells serveto trap individual live cells in well-defined spaces where they may bemeasured using mid-IR techniques. For example, a suspension of cells maybe flowed through the channel 1302, and stopped momentarily to allowcells to drop into wells 1304. The suspension and stoppage time must becoordinated such that the majority of wells contain a single cell.

FIG. 13b shows how the wells may be then scanned using mid-IR light 1008from one or more QCLs. Scanning may be accomplished by translating themicrofluidic chip, or the laser leading mechanism. As described herein,this may be accomplished in either reflective or transmissive mode. Thisscan may be repeated multiple times in the case where a time series ofmeasurements is being established to monitor changes in cells, withconditions such as temperature or chemical inputs through the channelpossibly being varied. Such time series may be used, for example, tomonitor cell differentiation (such as for stem cell applications), cellmetabolism for drug studies, and the like.

Using this design, cells may also be separated using optical orelectrical or combined techniques. For example, a visible or NIR beammay be used to address a well, and the pressure and heating from thisradiation displaces the cell resident in it back into the flow layer,where it may be flowed out of the cell. Electrical heaters in the wellmay do the same, or electrostatic forces may be used to separate thecell from the well. These operations may be performed in parallel. Forexample, a projector-type system may be used to project light intomultiple selected wells simultaneously, causing the cells containedwithin them to be pushed up into the flow region, and transported out ofthe device. This may open the potential for higher throughput processingusing large arrays.

FIG. 14 depicts another embodiment in which the microfluidic chamber maybe 2-dimensional. A 2-dimensional fluid chamber 1402 may be formedbetween the upper and lower caps. Optionally, if the area is very large,spacers 1404 may be inserted to maintain consistent spacing between thecaps. Cells flow into this area where they may be measured using one ormore QCL(s) 1102 and mid-IR detector(s) 1104. Measurements may beperformed either by translating the microfluidic chamber relative to thebeam 1008, or moving the beam itself from cell to cell. This may becontrolled by a human operator, or an image processing system may locatecells and steer the QCL-derived beam onto them for spectralinterrogation.

Alternatively, an “optical tweezer” beam where a laser spot 1408 (suchas visible or near infrared) or annular pattern may be used to trap andmove cells within the chamber 1402. This method, which is welldocumented elsewhere, may be used to immobilize moving cells, such assperm cells, or mobile microorganisms for interrogation by mid-IR. Itmay also be used to move cells to a specific measurement volume 1410. Inthis case, the mid-IR interrogation region may be stationary, and thecells may be translated in and out of the measurement volume using theoptical tweezers. The optical tweezers may be used to immobilize thecell temporarily, or at least confine it to the measurement volume forsperm cell inspection, for instance during the mid-IR measurement. Insome cases, such a visible or NIR beam may also be used to manipulatethe cell optically, by heating it, and puncturing its membrane, with theentire process observed by the mid-IR spectral system based on QCLs.

In an embodiment, the optical tweezer may be used to translate a cell ina scanning motion over the measurement region, in order to build up an“image” of the cell at a sub-cellular level. This method may be usedtogether with special optical features patterned on one of the caps,such as plasmonic or negative index structures that focus mid-IR lightto a subwavelength spot for high resolution interrogation of the cell.

FIG. 15 shows an alternate embodiment of the mid-IR optics combined witha microfluidic channel 1502 in a top view. In this embodiment, opticalwaveguides capable of carrying mid-IR light without high losses may beused to deliver light from one or more QCLs to the measurement volumewithin the microchannel. These waveguides may be fabricated from a rangeof mid-IR transmissive materials. For example, silicon may be bonded toa glass wafer and patterned into waveguides using standardphotolithographic techniques. In this example, an input waveguide 1508delivers the mid-IR light to the measurement volume containing a singlecell 1504, where it is absorbed according to its wavelength(s) and theconcentration of various chemical constituents in the measurementvolume, including the cell. The transmitted light may be collected bythe output waveguide 1510 and delivered to a mid-IR detector.Alternatively, a reflective design may be used to get two or more passesthrough the cell (with output to waveguide 1512)—more than onereflection may also be used.

The potential advantage of a waveguide-based system may be a veryconsistent alignment of the light source and output relative to themeasurement volume, and the beam shape (and therefore illumination)relative to the volume. Changes in beam location and profile relative tomeasurement volumes in the present invention may be calibratedinitially, before use, or periodically, such as through the use ofcalibrated particles, for example, tiny plastic spheres of knowndiameter flowed through the microfluidic measurement cell. In addition,the strong absorption of water and other liquids in the mid-IR range mayallow the system to be calibrated more easily using a liquid-onlyapproach.

FIG. 16 shows a system for the growth and purification of cells based onthe present disclosure. A unique aspect of the disclosure may be that itallows repeated interrogation of live cells without labels and UV lightor other methods, meaning the preparation steps (such as labeling) maynot be required, cell function may not altered, DNA may not be damagedsuch as that caused by some chemical stains and UV light, and noprocessing may be required to remove labels. For the aforementionedreason, this makes the disclosed approaches ideal for use in systemswhere cells or their descendants may be repeatedly interrogated for thepurpose of “guiding” growth or purifying a cell culture.

In an embodiment, an integrated bioreactor chamber 1602 may form thecenter of the system, where cells may be incubated and may multiply,differentiate (such as for stem cell cultures), or produce biochemicalcompounds of interest. Cells may be introduced to the system through acell input 1604. On the way into the reactor, the cells may pass throughthe measurement volume 1608 which may be interrogated by one or moreQCLs, and optionally visible/NIR lasers. During input, the cells mayundergo purification, time permitting, through the use of a cell sortingswitch 1610. Cells that do not meet certain criteria may be sent to theoutput well 1612 for disposal. Those cells that do meet the criteria maymove through the reactor input 1614 into the bioreactor chamber 1602.The bioreactor chamber may take multiple forms. For example, thebioreactor chamber 1602 may either be integrated into the microfluidicchip, or be implemented in macroscopic form. Multiple mid-IRmeasurement/sorting (1608, 1610, etc.) microfluidic devices may beattached to a single bioreactor in order to achieve higher throughput.The reactor may function in ways well known to those skilled in the art,with cell culture media/growth promoters, etc, and potentially surfacesor structures that may allow temporary attachment of cell structures.Media to feed cells and promote growth, differentiation or mutation or,to stress cells with particular compounds may be introduced to thebioreactor through an input 1620, and waste products may be removedthrough an output or exhaust 1622. In addition, temperature and otherconditions in the chamber may be controlled. In one embodiment, thecontrol is through a feedback loop based on system-integrated sensors.

After a specified interval or operations, cells may be extracted throughreactor output 1618 and may be flowed through the measurement volume1608 and cell sorting switch 1610. This process may eliminate cells notmeeting the criteria, such as by ejecting them into output 1612, andflowing the remainder back into the chamber through reactor input 1614.At the end of a specified interval or operations, a final sort canoutput the desired cells into the output 1612 for collection. This finaloutput can be different from previous sorts in which the rejected cellswere output. An example application is in the use of pluripotent stemcells to grow specific cells for implant into an organ. Initially, theinput cells may be sorted to place only pluripotent stem cells into thebioreactor. These cells can then be cultured in order to multiply, withcontinuous measurement and selection to remove any cells whichdifferentiate prematurely. Next, chemicals/media may be introduced intothe reactor and conditions changed that may promote differentiation intothe target tissue type. At this stage, the QCL-based sorter may serve tocontinuously remove cells that may have been differentiated into thewrong type of cell. Finally, at the output step, onlyproperly-differentiated cells may be sent to the output 1612, with anypluripotent cells removed to avoid potential tumor growths in the targetorgan. As such, the use of the output 1612 can change at differentphases of the process.

Another example where such a system is of interest may be in guidingevolution of cells or even small multicellular organisms with specificchemical properties. For example, the system shown in FIG. 16 may beused to evolve algae that may be efficient in producing precursors tobiofuels. In this example, a system based on the present disclosure,which may be used to characterize and measure intracellular chemicalconstituents with high throughput, and without damaging the cells, maybe of interest in performing high-throughput “guided evolution” ofoptimized algae cells. In this case, the bioreactor serves to multiplysuccessful cells following the introduction of a certain number ofmutations, and the QCL-based single-cell chemical measurement system andsorter then serve to measure levels of desirable products being producedby live cells (without puncturing them), and removing those cells whichdo not meet criteria. Such a system has the potential to eliminate slow,tedious work with macroscopic samples, and significantly acceleratedevelopment of optimized cell cultures. Similar methods may be used to“breed” cell cultures for use in other applications, includingpharmaceuticals, “green” chemicals, nutritional supplements such asomega-3 fatty acids, and other substances that may be produced bymicroorganisms.

FIGS. 17a and 17b shows two potential configurations for QCLs and mid-IRdetectors in the present disclosure. In FIG. 17a , multiple QCLs 1702may be combined into a single beam by mirrors, such as half mirrors, orthin film interference filters 1704. The use of wavelength-specificfilters reduces the losses associated with combining multiple beams, butmay reduce the flexibility of the system if multiple tunable QCLs may beused with overlapping wavelength ranges. The beams may pass through themicrofluidic measurement volume 1708 and may be collected by a singlemid-IR detector 1714. In this configuration, the QCLs may be modulatedin a manner that allows their signals minus absorption in themeasurement volume to be easily separated by electronic means in thedetector output, by methods well known to those skilled in the art. Forexample, where pulsed QCLs may be used, they may be alternately pulsedsuch that they result in discrete measurements on the detector.Alternately, they may be modulated at characteristic (and potentiallydifferent frequencies), and the signals separated at the output throughthe use of analog or digital frequency filters.

FIG. 17b shows a configuration where the QCLs are arranged in the samemanner, but there may be separate detectors 1714 corresponding to eachwavelength, as filtered by filters 1712. The potential advantage of thisconfiguration may be higher system bandwidth, and the use of narrowpassbands in filters 1712 to further reduce background signals in themid-IR.

FIG. 18 shows an embodiment of the present invention where it may beused to sort live sperm cells for the purpose of pre-fertilization sexselection. A sample 1802 may be provided to the system through an input1804. A filtering mechanism 1808, which may for example be a densemedium through which only sperm cells may travel is used to separatesperm cells from other constituents of the semen. The filteringmechanism may optionally include a centrifuge or pressure function. Thefiltering mechanism may optionally select for motile sperm. This stepmay be done externally from the system through methods well known tothose skilled in the art. Such a filter could consist of microfluidicfeatures.

The filtered sperm cells possibly with the addition of a medium whichpromotes flow and viability may be transported to a sorter input chamber1810. From this chamber, they may flow into the measurement volume of amicrofluidic channel 1812, such as through microfluidic features whichorient cells and prevent clogging. Multiple microfluidic channels andmeasurement systems may be used where higher throughput is required.When a sperm cell arrives in the measurement volume 1814, it may bedetected through the use of a visible or NIR laser 1818 and a scatteringdetector 1820. The scattered signal may be used to determine whether thecell entering the measurement volume is indeed a sperm cell, and whetherit is oriented correctly, and/or visibly malformed. The signal may alsotrigger the use of QCLs 1822. In an embodiment, two QCLs may be used.One QCL 1822 may be used to measure the asymmetric phosphate bondvibration at approximately 1087 cm⁻¹ which is characteristic of the DNAbackbone. Another QCL 1822 may be used to establish a reference level,which may be done with a wavelength just off the primary QCL, or at aknown reference wavelength that establishes a reliable baseline for themeasurement. One or both of the primary and reference QCLs 1822 may berapidly scanned in wavelengths over a narrow range in order tofacilitate measurement of a second derivative, a common signal used ininfrared spectroscopy. The QCLs 1822 may be used in pulsed mode toachieve higher peak power, and to allow use of a single mid-IR detector1828.

The reference wavelength may not be strongly absorbed by DNA or anyother target analyte. A subtraction, comparison or other analyticalprocedure may be done, such as by a processor, of the reference signalin relationship to the primary signal. Measurement of the signal itselfmay involve measuring an amplitude of absorption, standard deviationfrom the peak of the signal and/or reference curves, AUC/integration ofthe signal, and the like. In some embodiments, the reference signal maybe just the signal measured prior to an absorption curve.

A pre-filter 1808 may enable sorting cells by size, a surface propertysuch as an antigen or a cell surface protein, a chemical composition, acharacteristic determined by embodiments described herein, and the like.

Mid-IR light from the QCLs 1822 passes through the measurement volume1814 and any cells contained within it, and transmitted light may bemeasured by mid-IR detector 1828. The calculated absorption in the DNAband, as normalized by the reference QCL wavelength and measurementsbefore and/or after the cell passes through the volume may be used tocalculate total DNA contained within the sperm cell. This calculatedconcentration may be used to determine whether the cell is carrying X, Yor unknown chromosomes. Depending on the desired sex of the offspring,the cell is then either let through unchanged, or disabled/destroyedthrough the use of an infrared laser 1830. To disable a sperm cell, theinfrared laser 1830 may be pulsed at high power, focused on the cell orpreferably, the tail of the sperm cell, which may immobilize the spermcell. The use of a wavelength in the NIR (800 nm or higher) may minimizethe possibility of chromosomal damage, should a sperm cell treated insuch a manner still result in fertilization. Alternately, multiple,lower-power pulses of laser 1830 may be used. Subsequently, the spermcells, both viable and immobilized, may be transported to themicrofluidic output 1832, where they may be then filtered using a filter1834. This filter should ideally select for motile sperm cells through amethod such as the “swim-up” method, where sperm swim through a thickcultured medium, thereby enabling collection of only motile cells. Asmentioned hereinabove, microfluidic, or well-known macroscopic methodsmay be used to achieve this filtering. The output 1838 is then placedinto a delivery mechanism 1840.

In various embodiments, the sperm cells may be cryogenically preservedbefore use. In this case it would be desirable to preserve themimmediately after sorting, and then run a motility-based filter afterunfreezing, since the freezing process itself may render a portion ofcells immobile. The present disclosure thereby enables a sperm selectionsystem that does not expose sperm cells to harmful dyes or to UVradiation, that may achieve high specificity in terms of genderselection, and that may be implemented in a closed system, whicheventually makes it viable for small clinic or even home or farm use.

FIG. 19a shows a cross-section of a fluid stream or flow 1902 orientedto carry cells through a measurement volume such that flow is into orout of the plane of paper in this case. The flow is illuminated usingone or more QCLs, with a hypothetical illumination profile 1908 shown.Note that this may be an unlikely illumination profile and is shown toillustrate the invention only. Various beam shaping optics may be usedto shape the beam relative to the flow and relative to the region wherecells are expected to flow through the measurement volume. The mid-IRillumination is shown as 1904, illustrated in this example to be widerthan the flow 1902. It should be noted that a beam narrower than theflow 1902 may be used as well, and may be preferable from an opticalpower and contrast standpoint. The mid-IR light is strongly absorbed bywater in the flow 1902 and therefore, a characteristic pattern 1910 isseen in the detected transmitted radiation even in the absence of a cellin the measurement volume. The light may be more strongly absorbed inthe center since the cross section of the stream in this case iscircular. Ultimately, it will be highly desirable to measure thetransmitted mid-IR radiation using a single detector; however, this onlyresolves the total power transmitted and not the beam profile.

FIG. 19b shows the same configuration as FIG. 19a with the addition oftwo example cells 1912 and 1914 in the flow. The positions may beexaggerated for illustration, and two cells may be shown simultaneouslyonly to show positional variation over time. One cell 1912 is shownwell-centered in the flow, while another 1914 is off-axis. Thetransmitted intensity profile 1918 shows how this positional variationresults in different power levels observed by the detector. Each cell inthis example may cause the same incremental fractional absorption of IRlight. However, there is a difference in the pathlength through the flowbetween the two cells that is caused by the difference in theirlocations. The light that interrogates the cell in the center of theflow passes through a longer pathlength in the flow (typically mostlywater) than the light that interrogates the cell at the edge of theflow. As a result, the on-center cell 1912 causes a different intensityprofile at the detector than the edge cell 1914. This difference inintensity profile shown as fractional absorptions 1920 and 1922 forcells 1912 and 1914 respectively can lead to different relative changesin power detected by the detector depending on where the cell is locatedin the flow. Where the relative changes in power detected when cells1912 and 1914 pass through the measurement volume are shown in FIG. 19bas 1924 and 1928 respectively. If cell position in the flow causes thecell to block more or less of the mid-infrared light, the accuracy ofthe cell measurement will not be as high.

As a result of this position-dependence, depending on the positionvariation of cells in the flow, and on the signal ultimately beingmeasured, it may be desirable to either minimize effect of position bydesign, or compensate effectively for cell position as it flows throughthe measurement volume.

FIG. 20a illustrates one configuration of the present invention thatminimizes the effect of cell position in the flow that results fromwater absorption. In this case, a rectangular or square flow 2002cross-section may be used to provide equal path lengths through liquidfor all parts of the mid-IR beam. A rectangular channel 2004cross-section may be formed in material that is transparent to mid-IR atleast on the optical input and output sides. These surfaces may beantireflection (AR) coated for water to minimize stray reflections fromsurfaces and any resonant effects. The input beam profile 2008 may beused to illuminate the channel. In this example, the portions of thebeam outside of the sampling volume may be blocked, either by materialchoice or by making a mask that rejects this light (which would reducecontrast ratio in the measurements). As a result, the transmittedradiation 2010 may have a very consistent power profile spatially(limited by the input beam profile and diffraction effects).

FIG. 20b illustrates the rectangular channel with two hypothetical cellpositions in the measurement volume, a well-centered cell 2012 and anoff-axis cell 2014. In this configuration, the fractional absorptioncaused by the cells at different positions may be of the same amount oflight (same path length), as is illustrated by absorptions 2020 and 2022shown on the output intensity profile 2018. As a result the powerchanges 2024 and 2028 that are detected by the detector may be the sameregardless of cell position in the flow.

A square or rectangular flow cell may be implemented in a number ofways. Such a nozzle could be drawn using standard glass-formingtechniques and made out of IR-transmitting glassy materials such aschalcogenides glasses. However, probably the most proven way to buildsuch a channel may be by building a 2- or 3-layer structure with onelayer patterned to form a channel. For example, two wafers ofIR-transmitting material could be AR-coated (for AR in water on oneside, and air on the other), a material that may be readily patterneddeposited on one of them, channels patterned into this material, andthen the wafers bonded together and diced to produce multiplemeasurement channels suitable for the present invention. Multiplemethods for forming such microfluidic channels are known in the industryand may be used to implement the present disclosure. Of course, thematerials on optical input and output sides must be IR-transmissive. Forexample, Si, Ge, ZnSe and other materials with good mid-IR transmissionmay be used to form the channel that acts as the measurement volume.Optionally the material may also be chosen to transmit UV, visible ornear-infrared (NIR) light in order to make other measurements of theflow and cells including scattering, such as measurement of cellpresence, size, density and the like, and fluorescence, where acombination of traditional fluorescent label-based measurements andMid-IR measurements are desired.

FIG. 21 shows an embodiment of the present invention implemented in astandard flow cytometry system, in this case fitted for mid-IR cellmeasurements based on QCLs. The flow cytometric nozzle uses a sampleinjector 2102 to provide a steady flow of cells in a “core” flow, whichis combined with a sheath flow 2104 that surrounds it and merges with itin a laminar flow, which then narrows 2108 as it reaches the measurementvolume 2114 and ejection nozzle 2116. An alternative to the core/sheatharchitecture that is most commonly used may be the use of acousticfocusing of the cells into the center of a single flow. In manytraditional flow cytometers, the stream is interrogated using UV andvisible lasers while it is a continuous flow in air 2110. In someembodiments, such lasers may also be used to measure cell properties,both through scattering and optionally through standard fluorescentlabels. Due to pressure waves applied to the nozzle liquid, the streamthen breaks into individual droplets 2112, which may be important forcases where cells are sorted into populations. In an embodiment, a cellsorter case, charge may be applied to the sheath fluid just before eachdroplet breaks off, based on the measurements that have been done on itsvolume and any cell it contains. However, in other embodiments, aplurality of cells could be used. Electrostatic fields are then used toguide these droplets into two or more output positions, where they maybe collected or discarded. Such systems are in wide use, andwell-understood by those familiar with the field of cell sorting.

In an embodiment, the present invention adds use of one or more QCLs2118 and mid-infrared detectors 2124 to the cell measurements, fordirect chemical measurements through vibrational spectroscopy. Ratherthan measure the flow in air, where it should be circular to facilitatesmooth flow and break into droplets, the mid-IR measurement may be donein an enclosed measurement volume using a mid-IR transparent flow cell2114. This flow cell 2114 is made of mid-IR transparent material andAR-coated in order to reduce reflection losses and artifacts (AR coatedfor water internally, and for air externally). The QCL-generatedmid-infrared light 2120 may originate from one or more QCLs 2118, eachof which may be wavelength tunable at either high or low speed. Wherein,low speed wavelength tuning can be used for cytometer setup fordifferent tasks, in contrast, high-speed wavelength tuning, for examplethrough the use of drive current, can be used to do a spectral scan overa particular absorption peak of interest. The light transmitted 2122through the measurement volume 2114 and any cells contained within itmay be then sent to one or more mid-IR detectors 2124. Multipledetectors 2124 may be used to enhance the speed and/or signal-to-noiseratio (SNR) of the system through the use of one detector per wavelengthrange that is emitted by multiple QCLs 2118. For example, a simplesystem would employ two QCLs 2118, one for the signal of interest, andone for reference level. The light from multiple QCLs 2120 may becombined using a dichroic thin film filter or other wavelength-dependentcomponent, such as a grating, into a single input beam; similarly thetransmitted mid-IR light 2122 may be split using a thin film filter intotwo mid-IR detectors 2124, such as MCT detectors, one measuring thesignal wavelength and the other measuring the reference wavelength. Anenhancement to this base system may rapidly scan one or both of the QCLs2118 over narrow spectral ranges, allowing a local derivative and secondderivative in absorption to be measured, which can greatly enhanceaccuracy against a varying absorption baseline.

The measurement volume 2114 may be fabricated with round cross-sectionor preferably a square or rectangular cross-section to eliminate opticaleffects due to varying path lengths, as described hereinabove. The flatside walls provided in square or rectangular cross-sections can alsoreduce lensing and other optical effects that result from lightimpinging on transparent surfaces at an angle other than perpendicular.Both the external air-facing and internal liquid-facing surfaces may beAR-coated to reduce light loss and stray reflections, and minimize anyinterference effects. In this architecture and in the otherarchitectures described herein, the present invention may be combinedwith traditional optical measurements using UV, visible or NIR lasers orother sources to interrogate cells in the flow. For example, fluorescentlabels attached to the cells may be read using UV and/or visible lasers.The combination of traditional fluorescent label readout and mid-IRvibrational measurements may open up new possibilities for research andclinical applications. For example, fluorescent antibody labels mayestablish a “yes/no” measurement for a particular type of cell, andmid-IR measurements may then be used to establish quantitativemeasurements of biochemical concentrations of materials such as DNA, RNAand the like. Such a combined-mode measurement system may further beused to research and develop mid-IR measurement techniques that offerhigher accuracy, lower cell damage and/or easier processes with betterapplicability to clinical or field devices than their fluorescent labelequivalents.

Another example of combined modes may be the use of scatteringmeasurements to determine cell size and shape. Screening cells by sizeand shape, such as to determine the type of cell or the alignment ofcells, may be combined with mid-IR measurements of chemical constituentsto provide measurements of a particular cell population, or to providehigher accuracy measurement.

On the output side of the cytometer, there may be various options otherthan breaking into droplets and sorting as shown in FIG. 21. The cellsand fluid may flow directly into a waste container, keeping the systemclosed, wherever simple measurement is the objective. Where sorting ofthe cells after measurement is required, but a closed system isdesirable, a number of alternative sorting techniques have beendeveloped, including mechanical sorters where two or more channels areplaced into the flow as described previously and illustrated for examplein FIGS. 8 and 12.

Another technique that may be combined with the present invention toproduce a closed, “all-optical” cell sorting system is photodamage cellselection. In this architecture, after measurement has been completedusing mid-IR absorption measurements possibly in combination with moreconventional techniques, those cells that may not be desired in thepopulation are subjected to pulses of light, typically from a laser,sufficient in power to damage or destroy them. One example ofphotodamage is to use pulses of light to immobilize sperm cells, wherethe pulses of light are in the 1000-2000 nm wavelength range and therebythe photodamaged sperm cells are not able to fertilize an egg. Thisapproach of immobilizing the sperm doesn't damage the sperm's DNA. Inother cases, photodamage with a higher power laser pulse or a differentwavelength such as a UV wavelength can be used to puncture the cellmembrane and thereby destroy the cell to remove the cell from apopulation. Other non-optical techniques such as acoustic, mechanical,and RF have also been described to selectively damage cells and thesetechniques may be used in embodiments of the present disclosure.

FIG. 22 shows the present disclosure embodied in an architecture thatallows cell position in a flow to be measured accurately, in order tocompensate for position-dependent variations in mid-IR absorptionsignal. The flow 2202 shown in cross-section and directed in or out ofthe plane of the page contains cells 2204 to be measured using mid-IRlight 2210 from one or more QCLs 2208, the transmitted portion of whichmay be measured by one or more mid-IR detectors 2212. In someembodiments, the mid-IR beam is combined with one or more visible/NIRbeams 2218, from light source(s) 2214, which may be used to accuratelydetermine cell position and, potentially, size and shape. Dichroicfilters 2220 and 2224 may be used to combine and separate, respectively,the visible/NIR and mid-IR beams. When the visible/NIR light strikes thecell 2204, it may be partially scattered 2222. This scattering isdetected by visible/NIR detector(s) 2228. This configuration shows onlyforward scatter detectors, detectors may be used at other angles tomeasure wider-angle scattering. If an array of light sources and/ordetectors is used, they may be placed in a manner as to enhance theaccuracy of the position measurement, for example, they may be placed ina diagonal line across the flow in order to add a time component to themeasurement, as cells flow by the light sources/detectors.

The ability to accurately determine cell position in this manner mayallow the mid-IR absorption signal detected by detector 2212 to becompensated for cell position. In some embodiments, if the flow 2202 iscircular in cross-section and the cell 2204 is displaced from thecenter, the fact that more mid-IR light signal is originating from thepath containing the cell relative to when the cell is centered in theflow may be used to compensate the apparent absorption signal. Inaddition, visible/NIR sensors may be used to monitor the position andshape of the overall flow relative to the mid-IR laser light path andthe lensing effects provided by the shape and refractive index of theflow itself based on its position, diameter, and shape so that thesefurther effects can be compensated for in the mid-infrared measurements.

In all of the embodiments in this invention, it can be useful to comparemid-IR transmission signals before and after cells pass through themeasurement volume. This normalization is important because waterabsorption of light can be very strong in the mid-IR.

In systems such as that shown in FIG. 21 where the inner core and outersheath portions of the flow come from different sources, the materialsin the core may be different from those in the sheath. FIGS. 23a and bshow an embodiment of the present invention where additives in the coreor sheath fluid in a flow are used to create a “tracer” which may bemeasured directly by the mid-IR subsystem, and therefore allows accuratereal-time calibration/compensation for core flow position, andvariations in flow shape and position.

FIG. 23a shows a sheath flow 2302 surrounding the core flow 2304 beingilluminated using mid-IR light 2308 from one or more QCL(s). In thisexample, a tracer may be added to the core flow at a knownconcentration. The tracer may be selected to absorb at the wavelength ofone of the QCL sources in the system. If the core flow diameter may becontrolled accurately, such as through accurate core/sheath pressurecontrol, its absorption may then be used to calibrate mid-IR absorptioneffects, such as water absorption, on the core flow-related signal byusing readings before and after cells pass through the measurementvolume.

FIG. 23b shows the same flow with a cell 2310 in the measurement volume.In an embodiment, a configuration of this system may employ at least 2QCLs with different wavelengths, or 1 QCL that may rapidly wavelengthtune over an absorption peak shared by the tracer and the target cellcomponent. The concentration of the target compound in the cell may thenbe determined by measuring the absorption at the target mid-IRwavelength relative to water absorption without (FIG. 23a ) and with(FIG. 23b ) the cell present. This may give a signal relative to a knownconcentration, with flow-position-related factors removed.

FIG. 24 depicts another embodiment of the present invention in whichmultiple mid-IR beams are used to interrogate the measurement volume.The use of multiple beams allows the position as well as the absorptionof a cell 2404 to be measured more accurately, akin to computedtomography methods used in medical imaging. One or more QCLs 2408 emitmid-IR light that may then be split into multiple beams by multiplepartially-reflecting mirrors 2412. These beams, with the help offully-reflective mirrors 2414 may be then directed at multiple differentangles (3 angles are shown) through the flow 2402, which is shown incross-section and flows in or out of the plane of the page, and anycells 2404 contained within it. The beams are then detected usingmultiple mid-IR detectors 2410. Measurements made before or after a cellpasses through the measurement volume in this example may be used tonormalize the measurement, as they reflect the position and shape of thewater flow. Measurements of the cell made as it passes through thevolume may be from the multiple angles. By using multiple measurementsfrom different angles, plus comparing the measurements of the flow withand without the cell present, both the position and the absorption ofthe cell within the flow can be determined according to algorithms wellknown in computed tomography and according to techniques previouslydescribed herein.

In a yet further embodiment, the system may be embodied as amicrofluidic chip for measurement or sorting. Where, an example ofoperation of the chip may include procuring a sample of cells insuspension from a patient or culture, with any necessary protocol toindividuate the cells in the suspension; placing the entire sample intoan input port of a sample carrier, which can be a one-time-use plasticcarrier; and placing the sample carrier into a tool. Then, a cassettecomprised of multiple microfluidic chips may be inserted into the tool.The use of a cassette of multiple microfluidic chips allows for themicrofluidic chips to be changed during the process of measuring orsorting the sample, if clogging or degradation occurs.

Microfluidic chips can be built from materials that are opticallycompatible with the spectral cell measurement being performed. In thecase of mid-infrared (QCL) based measurement, the chip may be made ofmid-IR transparent materials including but not limited to Si, Ge, ZnSe,CaF2, BaF2 or salts with protective coatings. In addition, themicrofluidic chips can have structural or mounting features that can bemade from opaque materials such as structural plastics, metals orceramics. Surfaces of the chips may have functional coatings thatprotect them from fluid, are hydrophilic (to promote flow of liquidthrough measurement channel) and provide anti-reflective (AR)functionality. AR coatings may be applied to external surfaces tominimize reflections, and to the top and bottom of microfluidic channelsin the measurement zone in order to prevent reflections, maximizesignal, and minimize resonant optical effects (in this case the ARcoatings are designed to minimize reflection at the interface withwater). These internal (channel surface) AR coatings are particularlyimportant where a material with a high refractive index is used toconstruct the chip, as a resonant optical field could be created withinthe channel that would cause optical field intensity, and associatedinteractions with biochemical components of any cells being measured tobe dependent on the vertical position of the cell within the channel.Applying appropriate antireflective optical coatings on the surfaces ofthe channel reduces reflections at the interfaces, and therefore reducesresonance and associated variations in measurement accuracy due to thevertical position of cells within the channel.

In addition, the microfluidic chips may be pre-loaded with liquid in theinput and output volumes, as well as the measurement channel, toeliminate issues with starting fluid flow through the measurementvolume. Chips may include sealing layers/tapes on the input and outputreservoirs to maintain sterility, and prevent evaporation of primingliquid; the fluid in the input volume may be pre-seeded with particlesused for calibration of the chip. Chips may be charged with additionalliquid which is used to create a “sheath” flow around the core flowcontaining the cells; this same fluid may be used to apply pressure tothe system and maintain flow of cells through the measurement volume.

Known microfluidic configurations for creating 2D or 3D sheath/coreflows may be used. Sheath/pump fluid may be sealed in wells on the chipthat are opened for the purpose of pumping, or may reside below flexiblemembranes that can be addressed with an external pressure source,thereby maintaining fluid isolation.

The action of inserting the microfluidic chip into the tool opens theinput chamber of the chip, positions the chip onto the sample carrier,and moves a specific volume of sample onto the chip input reservoir.Where the system may be configured to provide continuous flow from thesample carrier to the chip, or charge the chip in specific increments,either one-time or in multiple batches. One potential architecture opensa tape on an input reservoir of the microfluidic chip, loads a smallvolume of the cell sample, re-seals the tape, and then moves the chip tothe optical measurement system, where it remains sealed until the samplehas been analyzed/sorted or until the chip clogs; then moves the chipback to the carrier, unseals the output reservoir, and transfers thesorted sample to the output reservoir of the carrier; the chip is thendisposed of (in the case where it is a measurement only, the chip may bedisposed of directly, without the transfer to carrier step).

At startup on a specific sample, the tool runs a small but statisticallysignificant sample of cells through the measurement channel of the chip,and into the non-selected (disposed) output reservoir. Each cell ismeasured using the vibrational spectroscopy system described herein. Forexample, it can be probed at three wavelengths corresponding to DNAabsorption, absorption of an interfering substance (which also absorbsat the DNA absorption wavelength; as an example certain proteins), and athird reference wavelength (to measure general absorption level due towater and other wide spectral features, for example). From these threemeasurements, a single DNA content number is calculated. The system alsomeasures the average throughput rate of the cells.

A histogram of cell counts vs. DNA content is generated and displayed tothe user. This gives the user an immediate sense for the distribution ofDNA content in cells, and whether there are distinguishable populations.It may give the user an immediate sense, for example, of whether apopulation of cells is multiplying (at this point some cells have twicethe normal amount of DNA). Optionally, the tool may “fit” Gaussian orother distribution curves to the observed population. These curves maybe application-specific (where a certain distribution of DNA content isassumed). Such curves serve to calculate expected purity in a sort ofthe cell population by DNA content.

The user may then select, through the user interface of the tool (whichmay be either on a built-in control panel, or on an attached devicessuch as a personal computer, laptop computer, tablet, smartphone, etc)at least two parameters of a sort, in the case where a sort is to beperformed, including the range of DNA contents to select for in the“selected” output sample, and the number of cells required in the outputsample.

With respect to the range of DNA contents that the method of theinvention could be used to measure characteristics of and select for inthe output sample, this could correspond, depending on the application,to significant cell populations including but not limited to: cells withX or Y chromosomes, where gender selection is being performed; cellswith an abnormal number of chromosomes, where cancer cells are beingseparated from a sample; cells that are in the process of dividing,where a cell study is being performed which involves looking atviability or growth rates (the inverse—those that are not dividing, mayalso be selected for); DNA content-based sorting may also be used in theprocess of separating differentiated from undifferentiated cells in stemcell related processes. If curves have been fitted to the populations,the tool will indicate the expected purity of the resulting sample asthe user moves the selection “window.” It will also adjust the expectedsort time for a desired number of cells in the output.

The method of the disclosure and the steps included as shown in FIG. 71will now be described relative to the system as illustrated in FIG. 24in conjunction with a processor to analyze the data and control theapparatus as an example. In Step 7101, the sample carrier containing thesample is loaded into the tool. In step 7102, the user chooses what todo with the sample, either measurement or, measurement and sort. Theuser also specifies the stopping criteria: either the number of cells orthe maximum sort time. With respect to the number of cells desired to beprovided in the output sample, the user may adjust this number, up to alimit set by the expected number of cells in the total sample, or by amaximum sort time. In the case of pure measurement, the number of cellsto be measured is selected. In the case of sorting, the number of cellsto be collected in the output sample is specified or; optionally thenumber of cells sorted may be set by requiring a certain sample sizewithin a specific DNA content window, in order to obtain a statisticallysignificant sample size.

In step 7104 the user specifies the measurement including wavelengthsfor measurement and aspects of the absorption or transmission spectrumassociated with the desired characteristics of the cells or particles tobe measured. The sample containing the cells or particles in suspensionis then loaded from the sample carrier into the microfluidic chip inStep 7106.

The user then starts the process in step 7108. The tool then initiatesthe measurement or sort. Cells or particles are flowed through themeasurement channel on the specialized microfluidic chip in Step 7110,at a rate and dilution that allows sufficient measurement time in themeasurement volume, and also allows the cells or particles to beaccurately sorted with the on-chip sorting mechanism. For example thefollowing steps may be performed by the system based on mid-Infraredquantum cascade lasers measuring mid-IR vibrational absorption featuresin the cells of interest.

Prior to a cell entering the measurement volume in Step 7112, the systemcontinuously measures the absorption of the (for example) three mid-IRwavelengths being used; the signals are generated by three QCLs that aremodulated in a manner such that their signals may be separated afterdetection by a single detector, such as a thermoelectrically (TE)-cooledmercury cadmium telluride (MCT) photodetector. The signals observed atthe detector before (and after) the cell passes through the volume areused as a baseline for the measurement, in order to cancel out, in largepart, slow-speed variations in the system, including variations inrelative laser power, changes in background absorption in the flowchannel, and slow-speed changes in optical effects in the system andchip (for example, mechanical changes or index of refraction changes dueto temperature). Modulations applied to these lasers may be of varioustypes. Pulsed modes may be used to generate short, distinct peaks, whereindividual wavelengths are pulsed in rapid succession. In continuouswave (CW) operation, the lasers may be modulated with specific carrierfrequencies that may then be separated using analog or digital filtersat the detector. Aside from power level modulation, the lasers may bemodulated in terms of wavelength. For example, wavelength may “chirp” aslasers are pulsed or modulated, because of current and temperatureeffects. This may effectively broaden the range of wavelengths at whichabsorption is measured. This “averaging” effect may be beneficial sinceabsorption features in liquid phase are typically quite broad. Externalwavelength-modulation components may be used within the QCLs to morebroadly sweep wavelength. For example, external cavity Fabry-Perotconfigurations may be used with wavelength-setting elements such asdiffraction gratings or tunable etalons. In one case, very rapid tuningover a broad range could enable measurement of both baseline (“valley”in the absorption spectrum) and signal (“peak” in spectrum) using asingle laser for a particular analyte. Center wavelength of the QCLs (orslave lasers, in the case of a CARS system), which may be set usingexternal tuning mechanisms or simply operating temperature (effected bya TE cooler on which the QCL is mounted) may be set to match theabsorption peaks (or valleys) observed in the cell sample. This processis performed during the setup of the system, and may be repeated atspecific intervals (during which all cells are sent to the “discard”output), or when the system senses recalibration is required. Thisprocess may be performed by sweeping the wavelength slowly as cells aremeasured, accumulating a distribution of signal points vs. wavelength,and then determining where the minimum or maximum signal occurred, andusing this as the wavelength set point.

As a cell enters the measurement volume, it is detected using avisible/NIR/SWIR beam that is scattered by the cellular material in Step7114. This beam is separate from the mid-IR beam and is used to detectcell presence, flow speed (along with other sensors, possibly), andunusual scattering signatures that could signal cell agglomerates. The“presence” signal generated by this beam may also be used to triggerintegration of the mid-IR signals measured by the system. In the casewhere the system is built with mid-IR lasers, the material of the chipmust primarily be transparent to mid-IR wavelengths. Some materialsoptions such as silicon are transparent in much of the mid-IR, but nottransparent in the visible regime. In this case, a SWIR laser (1.5microns, for example) and detector combination—such as those developedfor telecom applications—may be used to measure this cell presence andscattering. Cell presence and scattering may be measured intransmission, reflection, or both.

In the Step 7116, the cell or particle is measured with mid-infraredlight. Within the measurement volume, mid-IR light is transmittedthrough the cell or particle, where it is absorbed according to it'scharacteristic wavelength absorptions and the chemical constituents(chemical bonds) within the cell or particle. For example, where threewavelengths are used to make an accurate DNA measurement in a cell:there may be a rise in the detected signal when a cell is present (i.e.lower absorption of) due to the fact that the materials other than waterthat make up the cell, such as proteins, lipids, and DNA, displace waterand they have a lower absorption in the mid-infrared than water; whenadjusted for this change in background, a signal corresponding toproteins will show higher absorption; and when adjusted for the changein background, and after subtracting any known interferences fromprotein (calculated by the previous measurement), the DNA absorptionsignature rises.

The measurements may be integrated over the time that the cell resideswithin the measurement volume, and the integrated signals may be used toquantify cellular DNA. Other quantities (such as protein content, orcell size based on scattering) may also be measured, and may optionallybe presented to characterize the cell sample, or refine sort parameters.Combinations of DNA and protein content, DNA and lipid content, andother 2-axis or multi-axis analyses and sorts may be performed byappropriate extensions of the present invention, using appropriatemeasurement wavelengths for specific molecular bond vibrations.

Based on the information provided in Steps 7102 and 7104, in Step 7118the cell is sorted, wherein a decision is made either to allow the cellto proceed to the “discard” output reservoir of the chip, or be divertedto the “collection” output reservoir. The microfluidic channels in thechip may be configured so as to make the default route to the “discard”reservoir, and only when the switch is actuated will cells be able toreach the “collection” or “select” reservoir.

At a time interval after measurement, determined by the flow velocity inthe channel, a microfluidic switch may be actuated in order to push thecell into the appropriate output channel. At the position of thismicrofluidic switch, or somewhere along the microfluidic channelpreceding it, there may be another measurement point illuminated byvisible/NIR/SWIR light where cell presence is measured by scattering.This allows realtime measurement of flow/cell velocity in the channel,and allows the system to recalibrate timing of the microfluidic switchactivation.

The cell, which may have had it's position in the flow laterallyperturbed, is then routed into one of two or more output channels basedon the microfluidic switch action. There may be additional opticalmeasurement points in these output channels that serve primarily toverify that the switch actuation is functional and timing is correct,and observe any sorting errors that occur. Should errors occur, thesystem may adjust timing or magnitude of switch actuation, or simplydiscard the current chip and replace it with a new chip.

Cells are accumulated in at least two output reservoirs, the “discard”reservoir and the “select” reservoir. These reservoirs can be located inthe microfluidic chip or in the tool that the chip is mounted in.

During the sort, a number of different problem/failure conditions may bedetected by doing a preliminary analysis of process data in Step 7120,including but not limited to: sample out, or front-end clogging, flowrate variation, switching failure and cell density variation. If aproblem is detected, in Step 7122 appropriate action is taken asdescribed below.

With respect to sample out, or front-end clogging the condition isdetected by no additional cells appearing in the measurement channel.This can be caused by the input sample being exhausted, or the input ofthe channel becoming clogged. Following the detection of this problem,the system may: perform anti-clogging procedures, which could includerapid pressure pulses on the input or back flushing of the system;and/or move to unload and discard the current chip and load a new one tomatch up to the sample carrier as in Step 7106.

With respect to flow rate variation, through the use of opticalmeasurement points, the system may detect that the sample flow rate isout of bounds. In this case, the system regulates the pressure in thesystem to adjust the speed of cells in the measurement channel. The flowrate may be estimated from a single sensor at the measurement point (bymeasuring how long the cell is present in the beam) or by 2 or moreoptical sensors along the measurement channel (by measuring time a celltakes to travel between points). If the cell speed is out ofbounds—which may cause DNA measurement SNR problems, or switchinginaccuracy problems, the system may send all cells to the default“discard” reservoir until the speed is properly regulated.

With respect to switching failure, through the use of opticalmeasurement points on the output channels, the system may detect thatcells are improperly switched to one of the output channels. In such acase, the system may be paused immediately (if a very high purity sortis being performed). Switch timing relative to measurement may beadjusted, as well as magnitude of signal being used on the cell switchmechanism.

With respect to cell density variation, the system may detect that cellsare arriving with spacing that is either too dense—not allowing cells tobe switched reliably—or too sparse—prolonging the sort operation for agiven number of cells. The system may have provisions for adjusting thisdensity (such as dilution or concentration steps), or it may simplyupdate the user as to the change in anticipated purity or sort time.Dilution of cells may be performed during the transfer of sample fromthe sample carrier to the measurement chip; if this is the case,dilution may be adjusted from chip to chip until an optimum is found. Ifthe dilution or concentration required, to obtain the desired celldensity through the chip is excessive, a chip may be discarded, andanother loaded onto the sample carrier where upon, the process returnsto Step 7106.

When the sample loaded into a particular microfluidic chip has beensorted completely as determined in Step 7124, the chip's “select” outputreservoir is opened by the system, and its contents are transferred tothe output reservoir of the sample carrier in Step 7125. This may bedone with a liquid purge run through the chip or pipette action.

Once the chip's reservoir has been emptied, it is sealed, removed fromthe carrier, and discarded, potentially into a sealed compartmentattached to the disposable sample carrier. Provided the sample carrieris not empty as determined in Step 7126, a new chip is then loaded inStep 7128 and the process returns to step 7106.

This process continues until the sample carrier is determined to beempty and the sort is complete. At this point, in Step 7130 the user issignaled, and the sample carrier is prepared for unloading. The samplecarrier is removed from the tool, with the sorted sample ready for usein the output reservoir. The sorted sample may subsequently be removedby pipette or other method and used in subsequent lab or clinicalprocedures. A report is generated that includes the measurement data forthe cells and the related process data gathered during the measurementor, measurement and sort.

FIGS. 25a and b show two example configurations of the microfluidic chipsystem. FIG. 25a shows a tool that may accept microfluidic chips thatmay be pre-loaded by a user with the cell sample to be measured and/orsorted. This may be generally a configuration for relativelysmall-volume samples that may be contained on and sorted with a singlemicrofluidic chip. A slot 2502 may accept the chip into themeasurement/sorting system. A display 2504 shows histograms and otherindicators regarding the process or the measured characteristics of thecells or particles. The display may be a touchscreen display that mayallows the user to select process conditions, input sort set points, setthe graphical display of data, and start and stop operation. The toolmay include other interfaces, such as a USB interface to allow transferof data to (or control by) a computer, or transfer or backup of dataonto a USB memory stick. Wireless networking interfaces may be includedas well.

FIG. 25b shows another configuration of the tool described herein whichcan handle larger sample volumes. Again, a display 2504 may allow theuser to select process conditions, make setpoints and review data fromthe cell measurements and/or sort. In this configuration, however, thesample containing the cells or particles is loaded into a samplecarrier, which is loaded into sample slot 2508. Separately, a cassetteof multiple microfluidic chips, one or more of which may be used tosort/measure portions of the sample loaded in the sample carrier, may beinserted into chip cassette storage 2510. In this manner, multiple chipsmay be used, if needed, to sort a single larger sample loaded into thetool.

FIGS. 26a and b show example displays of measured data that can be shownto the user of the tool in a generated report, in this example the datais DNA content for sperm cells. FIG. 26a shows a configuration where DNAvs. cell count may be displayed in a histogram format. The x-axis 2602in this example may represent the DNA content of the cell. The y-axis2604 represents the cumulative cell count in the sample. Thedistribution of cells measured shown in FIG. 26a includes a distributionof cells in various stages. The peak 2608 with greater DNA content isrepresentative of cells that are actively dividing. The middle peak 2607is representative of cells with a normal DNA content. The small peak tothe left 2605 is representative of a small percentage of cells showingan abnormally low DNA count which could indicate aneuploidy. Thishistogram in itself may be valuable to the user for a rapid assessmentof the cell sample. In addition, curve-fitting algorithms may be appliedmanually or automatically, either in real time or offline after themeasurement to estimate the percentage of cells in each state.

FIG. 26b shows a configuration where an additional parameter besides DNAmay be used to classify cells. In this case, the x-axis 2610 againrepresents the cellular DNA content. The y-axis 2612, however,represents another parameter measured by the system. Examples of thisparameter may include, but are not limited to, secondary vibrationalspectral measurement of the cell using the same technique but differentwavelengths to determine for example, protein content, lipid content,sugar content, RNA content. Other examples of this parameter may alsoinclude visible/NIR/SWIR light scattering from the cell, possiblyindicating size and/or morphology. Examples of this parameter mayinclude shape, size and density parameters calculated from imagery ofthe cell in visible/NIR/SWIR wavelengths. Further examples of thisparameter may include fluorescence signal from dyes or labels, suchlabels could include but are not limited to dye for assessing cellviability through membrane integrity, membrane-staining dye to measureoverall membrane, antibodies attaching to specific cell types, and thelike. Yet other examples of this parameter may include quantum dot andother labels which function in a similar manner to fluorescent labels,though readout method is different. Yet further examples of thisparameter may include multiple other cell measurement methods known tothose in the field. It should be noted that this is not restricted toone additional parameter. Multi-dimensional cell classification may besupported by the present disclosure.

The density plot 2614 shows the cumulative density of cell counts in thesample. This may be represented by a simple scatter (dot) plot, andsupplemented with color or iso density lines to indicate statisticaldensity. In this example, there are two levels of DNA (for example adividing population of cells), and then two levels along the otherparameter. For example, if the other parameter here measured membranelipids as measured by a secondary vibrational signature, the plot mayindicate that there are two sizes of cell or cell agglomerates where twocells are adhered to one another while passing through the measurementvolume (and therefore should be rejected from the data, or sorted out ofthe sample).

FIG. 27 shows an example of a disposable measurement unit 2700,consisting of a microfluidic chip 2704 and carrier 2702 that may be usedin a system such as the one shown in FIG. 27a . In this disposablemeasurement unit, the microfluidic chip 2704 is embedded in a carrier2702 which includes an input port/reservoir 2708 and an outputport/reservoir 2710. For measurement-only applications, the output port2710 can be internal to the carrier 2702 so that the sample is disposedwith the disposable measurement unit following the measurement.Likewise, in sort applications, a single output port 2710 may beapplicable if a second output reservoir internal to the carrier 2702 isprovided for the rejected cells or particles so that they can bedisposed of together with the disposable measurement unit. The inputport 2708 may be a well into which a sample is pipetted, alternativelythe input port 2708 could be connected to a sample carrier with a largervolume.

Where optical measurements of the cells or particles is made along themicrofluidic channel, a window 2712 may be formed in the carrier 2702.The carrier 2702 can be made of plastic, ceramic or metal materialswhere it's primary function is to provide structural support of themicrofluidic chip 2704 along with some channels or reservoirs for thesample to flow through. By providing a window 2712 in the carrier 2702,a clear optical port can be provided for the measurement. This iscritical in particular in the infrared, where plastic may have very lowtransmission or may be opaque. The microfluidic chip or the window maybe manufactured of Silicon or Germanium, which may be transmissive inportions of the mid-infrared, and antireflection (AR) coated to minimizelosses and fringe effects.

The sample may be charged in the input port 2708, and then thedisposable measurement unit 2700 is inserted into the tool. In anembodiment, tubes to control pressure could then be attached to theinput port 2708 and output port 2710 in order to control the pressuredifferential and therefore the sample flow rate through the measurementchannel. The carrier 2702 may have features to provide some alignmentwithin the system. Alignment of the microfluidic chip 2704 with theoptical readout system may further be refined by passive or activemeans. For example, the microfluidic chip 2704 may havephotolithographically-defined mechanical features which allow passivealignment of the chip 2704 with the optical readout system. Alternately,the microfluidic chip 2704 may have photolithographically-definedoptical features which may be optically interrogated in order toactively align the chip to the optics or the optics to the chip 2704. Ina further embodiment the optical system performs a “search” in which ituses the inherent absorption signals of the measurement channel, thewater in the channel, and any cells flowing through the channel in thechip 2704 in order to optimize focusing and x-y position of the beamrelative to the channel.

After the measurement or sort is performed, the disposable measurementunit 2700 may be ejected from the tool. If undesired portions of thesample such as rejected portions of the sample, remain in the disposablemeasurement unit 2700 after use, the unit 2700 may be ejected from thetool into a built-in disposal bin appropriate for biohazardous samples.If the disposable measurement unit 2700 contains desired portions of thesample after measurement or sort, in reservoirs (2710 or other)associated with the unit 2700, the unit 2700 may be moved into an outputbin or storage area. The user may then remove the desired portions ofthe sample from the disposable measurement unit 2700 and proceed withthe appropriate protocol.

FIG. 28 shows an alternative configuration for a measurement unit 2800that can be used with the tools shown in FIGS. 25a and 25b , where acarrier 2802 is used together with one or more microfluidic chips 2804,where the chips 2804 may be separate pieces from the carrier 2802. Thisenables the use of multiple microfluidic chips 2804 thereby providing anumber of advantages including: higher throughput, redundant chips whereclogging or other chip problems are an issue, where precise calibrationof dilution is an issue, or where it may be desirable to run multiplemeasurements on multiple chips in parallel. In this case, multiple chipsmay be loaded with sample from the carrier, and run in parallel.

The measurement unit 2800 may have an input reservoir 2808 and zero ormore output reservoirs 2810. In this case where only measurements aredone on the sample, the output reservoirs may be internal to the carrier2802 and the measured sample is discarded with the carrier. One or moreoutput reservoirs 2810 or output ports may be used in cases where a sortis performed. This example shows a single output reservoir 2810 wherethe sample is available to the user after measurement, but multipleoutput ports or output reservoirs are possible.

One or more microfluidic chips 2804 may be provided in the carrier 2802,so that a portion of a sample can be induced to flow from the inputreservoir 2808 to the input port 2812 associated with the microfluidicchip 2804. The carrier 2802 may have compliant gaskets to allowefficient matching with the chip 2804, and a good seal around thetransfer locations. In this example, the measurement is then done inplace (on top of the carrier). A pressure differential may be applieddirectly to the input reservoir 2808 and the output reservoir 2810 toinduce the sample to flow into the input port 2812, through themeasurement volume 2818, into the output port 2814 and then into theoutput reservoir 2810 as it flows through the chip 2804. As the sampleflows through the measurement volume 2818 it is interrogated using thevibrational spectroscopy system. A window 2820 in the carrier 2802 maybe provided for clear optical access to the measurement volume 2818 andthe microfluidic channel.

If clogging or other problems associated with the microfluidic chip 2804are detected, the microfluidic chip 2804 may be removed and disposed of,and a new chip 2804 is matched to the carrier. This disposal may be doneat a regular interval to preempt clogging or other issues, and/or toeffectively charge a “fee” per incremental sample measured or sorted. Inan embodiment, the carrier 2802 includes a disposal chamber for themicrofluidic chips 2804, so that the system remains as closed aspossible, and consumables may be disposed of after each sample run.Similarly, the microfluidic chips 2804 may be included in the carrier2802, and removed from an internal magazine in the carrier 2802 asneeded. In this configuration, all consumables related to the processare delivered in a single carrier 2802 or cartridge.

FIGS. 29a and b show some example formats for microfluidic chips thatmay be used in the present invention.

FIG. 29a shows a microfluidic chip 2900 that could be used in ameasurement-only application. An input port 2902 may be used tointroduce the liquid sample containing cells or particles. Amicrofluidic channel 2910 transports this sample through the measurementvolume 2908 to the output port 2904. Features may be built into the chip2900 to prevent clogging at the entrance to the channel from the inputport. Features in this region, and within the channel itself, may alsobe used to orient the cells in a specific manner, such as to preventclogging, or to promote better measurement. For example, a pattern ofposts of specific size and shape may be used to break up agglomerates ofcells, or to block large agglomerations of cells or other substancesfrom reaching the channel. Certain configurations of posts may in factbe used to pre-select cells of a certain size for measurement in thechannel.

The chip 2900 may have photolithographically-defined mechanical oroptical features which assist in aligning the chip 2900 to the opticalinterrogation system. For example, an etch process alignedphotolithgraphically to the measurement channel (possibly the same etchprocess step as the one used to create the input and output ports), maybe used to create features for passive alignment of the chip 2900 to theoptical interrogation system. Alternatively, these may be opticalfeatures (reflective metal, or windows in a metal film) that allow thesystem to actively align the chip 2900 to the optics. Fine alignment maybe done with features in the measurement volume 2908, using the opticalsystem itself to optimally align for measurement.

FIG. 29b shows a similar microfluidic chip 2950, this one for use insorting cells or particles. A sample may be introduced into the inputport 2914 and flows through a microfluidic channel through themeasurement volume 2922 and microfluidic switch 2924 and then into theselect ports 2918 or the discard port 2920. After measurement in themeasurement volume 2922, characteristics of the individual cell orparticle can be calculated. Based on these measurements, and sortingparameters entered into the system, the cell or particle is routed toeither the discard port 2920 or the select port 2918. Routing may beperformed using a microfluidic switch 2924 or two pressure ports 2928may be used to shift the flow in the channel to one side, causing cellsor particles to move into one branch or another of the output junctionleading to either the select port 2918 or the discard port 2920, themechanism is described in more detail in FIG. 32 below. The microfluidicchannels may in some embodiments, be defined such that a “default” routeis established to the discard port 2920, and only when an actuation isperformed are cells routed to the select port 2918.

FIGS. 30a and b show additional example configurations of microfluidicchips for use in the present invention.

FIG. 30a shows a configuration of a microfluidic chip 3000 where adiluting/sheath fluid is provided along with the sample in order toprovide a centered flow of the sample in the measurement channels. Thesample may be introduced into input port 3002. A diluting fluid may beintroduced, or pre-charged, in port/reservoir 3004. This fluid may beused to apply pressure via a duct on the input port 3002 to drive samplethrough the measurement channel. In addition, side channels 3010 may beused to form a sheath flow around the core/sample flow at junction 3012.This centers the sample cells or particles in the flow as they passthrough measurement volume 3014. The output port 3018 then receives thesample as well as the sheath fluid. This configuration provides a numberof advantages: cell flow rate and spacing may be better controlled;cells are centered within a wider channel, where they can be measuredwith better repeatability. In addition, background signals from thesample fluid can be reduced in this configuration if the material of thesheath flow is chosen such that it has less effect on the backgroundsignal than the material that the sample is suspended in the core flow,as the core fluid flow may be typically very narrow compared to thesheath flow.

FIG. 30b shows a microfluidic chip 3050 configuration where cells orparticles in the sample may be switched, based on the vibrationalspectroscopy measurement, using an electric field at the switch point3032. Cells or particles flow from the input port 3024 through themeasurement volume 3028 where they may be measured as previouslydescribed. Based on measured individual characteristics of the cell orparticle, a voltage is applied to contacts 3030 which perturbs the pathof the cell or particle, and causes it to flow to a selected outputreservoir 3034.

FIG. 31 shows an example construction of a microfluidic chip 3100. Inthis example, the chip 3100 is constructed for use in a mid-infraredspectroscopic cellular DNA measurement system utilizing quantum cascadelasers as the optical source(s). A top wafer 3114 and bottom wafer 3102may be used. These wafers may be made from materials that aretransmissive at the mid-infrared wavelengths of the QCLs to be used forcellular interrogation. For example, float-zone Silicon, which has hightransmission in the infrared, and which is readily obtained andmachined, may be used. Other materials that may be applicable includeGermanium, ZnSe, CaF2, BaF2, and other materials well known in infraredoptics.

The bottom wafer 3102 may be first antireflection (AR) coated 3108 and3104. This is important for a number of reasons, such as to minimizelosses of IR light in the system and thereby maximize signal-to-noiseratio, or minimize QCL power required, reduce fringing artifacts frominterface reflections which may distort the transmission spectrum of thesample and therefore distort apparent sample absorption and,importantly, minimize any resonant optical cavity effects in the channel3112. If the channel becomes a resonant optical cavity, the fieldintensity may vary significantly with vertical position in the channel.In this case, the system may become sensitive to cellular position,because the cell may be in a position to absorb more infrared radiationat field maxima, and less at minima. This may result in higher and lowerapparent infrared absorption based on cell position, which should beavoided.

An alternative or complementary solution may be to create a flow inwhich cells are confined to a specific layer, and therefore aresubjected to the same optical field from cell to cell. A good AR coatingmay be a more robust and simple solution, and design of mid-infrared ARcoatings are well known to those skilled in the art. It may also beimportant in such a coherent laser optical system to reduce coherentoptical effects from the source itself, which cause position-dependencewithin the measurement channel. This is described in more detailelsewhere in this disclosure.

In an embodiment, internal AR coating 3108 may be designed so as toprevent reflection between the lower substrate material 3102 (usuallyrelatively high index) and the liquid sample in the channel(approximately the index of water, which is relatively low). Theexternal AR coating 3104 should be designed for the atmosphere of thetool, which may be most likely air. The coatings 3104 and 3108 must bedesigned to withstand subsequent processing and exposure to the sampleand associated fluids, without toxicity to the sample. For this purpose,very thin terminal layers may be provided on the internal AR coatings3108 and 3120 for both the bottom wafer 3102 and the top wafer 3114respectively.

The microfluidic features such as channel 3112 may be fabricated eitherby etching the substrate material 3108 to form a channel of specifieddepth (before AR coating), or, as shown here, formed through addition ofanother material 3110. For example, SU-8, a UV-crosslinked photoresistthat may be etched with high aspect ratio, and is known to bebiocompatible, may be spun on the bottom wafer 3102, and then patternedwith microfluidic features. The top wafer 3114 is first patterned andetched to provide ports/reservoirs and any mechanical alignment featuresindicated here by 3122. For this purpose it is desirable to use a wafermaterial where etch processes are well known, such as silicon. Internaland external AR coatings 3120 and 3118, and respectively, may bedeposited to the top wafer 3114 after this etch step or, if depositedbeforehand, removed during the etch process.

The top and bottom wafers 3114 and 3102 may be made from differentmaterials according to processing and operating requirements. Forexample, the top wafer 3114 may be made from silicon, which may bereadily etched using well-established processes, and may be a low-costmaterial in the mid-IR. Silicon, however, suffers from a number ofdrawbacks for mid-IR spectroscopy. First, it may have some absorption inthe mid-IR which may be mitigated by using high-purity float zoneSilicon. It may also not allow for visible or NIR light transmission,which may be desirable where visible light measurements may complementthe infrared cellular DNA measurement. For example, visible imaging,fluorescent label measurement, or other techniques may require amaterial with visible-light transmission. In such a case, it may bedesirable to use a material such as ZnSe, BaF2, CaF2 or other knownvisible/mid-IR window as the bottom wafer 3102, and Silicon as a topwafer 3114.

The top wafer 3114 and bottom wafer 3102 may then be aligned and bonded.A thin layer of SU-8 may be used for example, to establish a bondbetween the top 3114 and bottom 3102 wafers at the appropriatetemperature and pressure, usually under vacuum. This may be done with asupplemental layer or pattern of SU-8 on the top wafer, but some groupshave been successful with one-sided SU-8 bonding. In a furtherembodiment, multiple microfluidic chips are formed from larger sheets ofmaterials that are multiply patterned for top wafer 3114 geometries andbottom wafer 3102 geometries that are then aligned and laminated toprovide a mother wafer laminate that is subsequently diced into multiplemicrofluidic chip 3100 components. These may be individually packed intocarriers, or matched with carriers in the tool, as described earlier.

FIG. 32 shows the detail of an example embodiment of a microfluidicchannel in a top view with flow from top to bottom, including ameasurement volume and a pressure-actuated cell switch. The example isillustrated for the case where a mid-infrared, QCL-based optical systemmay be used for cell interrogation. An incoming channel 3202 carries acore stream with cells 3208 within a sheath fluid 3204 into themeasurement zone 3210. The measurement zone may be delineated using ametal mask that allows the light beam (indicated by dotted line 3212) topass only through the aperture 3211. In this embodiment, the aperture3211 is shown larger than the beam 3212, so that mechanical movements donot translate into large variations in the beam 3212. The aperture 3211,patterned photolithographically onto the microfluidic chip, may serve toallow alignment of the measurement beam with the center of the channel3202. The beam 3212 in this case, can comprise visible/NIR/SWIR lightalong with mid-infrared light.

As a cell or particle passes through the measurement volume 3210, itbreaks the visible/NIR/SWIR portion of beam 3212, indicating cell orparticle presence, and possibly giving some data on the size/density ofthe cell or particle. The mid-IR portion of beam 3212 may be absorbed bythe cell or particle according to its chemical bond constituents. Themid-IR portion of beam 3212 may have one, two or more individualwavelengths acting as reference levels, or to measure variousconstituents of the cell or particle other than DNA. For DNA measurementof cells, vibrational fingerprint regions such as the 1234 cm⁻¹ or 1087cm⁻¹ phosphate bond vibration lines may be used to measure absorptionand therefore the amount of DNA present.

Based on the determined characteristics of the cell or particle, a sortdestination for the cell may be determined. By default, the cellcontinues straight along the channel to the waste output (left branch).If the cell is determined to be of the sought-after type destined to the“selected” output, as it passes through the detection point 3218, thepressure actuating channels 3214 may be used to slightly offset the coreflow to the right. A visible/NIR/SWIR beam 3220 may be used in thislocation to accurately assess cell speed in the channel (from the delaybetween the measurement point and the switch actuator point). This speedmay be used to regulate the overall pressure in the system to maintainflow rates within a window, and may also be used to adjust the timing ofswitch activation relative to the cell passing through the measurementpoint.

By default the core flow may go straight into the “discard” channel asshown by portion 3228. Where the switch has been actuated to select acell, the core flow may go to the right of the branch point 3222 andselected cells 3224 may go to the “selected” out port/reservoir. Twoadditional optional detection points 3230 may be used in the discardchannel and the select channel with visible/NIR/SWIR interrogation tomonitor cells flowing into channels, so that errors may be detected andcorrected. For example, if sort failures are detected, the timing of theswitch actuation or the pressure on the system (and therefore cellvelocity) may be adjusted to provide accurate switching.

As is illustrated in the FIG. 32, cell agglomerates may be detectedeither by the visible/NIR/SWIR scattering or imaging patterns, or by theinfrared quantification. These may be sent through to the “discard”channel. Likewise, portions of the sample flow where cells may be spacedtoo closely within the channel so that accurate switching may not occur,may be let flow through to the discard, depending on the systemparameters. There are of course situations where very rare cells arebeing collected, in which case it is better to err on the side ofswitching cells into the collection reservoir.

Through a combination of flow speed monitoring and cell timingmeasurements provided by the aforementioned optical measurement points,the dilution of the cells may be monitored. If the cells areinsufficiently diluted, core vs. sheath pressure may be varied to spacecells, or the sample itself may be diluted differently by the tool,sometimes necessitating use of a new microfluidic chip.

FIG. 33 illustrates an embodiment of the optical interrogation system ofthe present disclosure. This example may be based on two QCLs 3308 atdifferent wavelengths. For example, one QCL 3308 wavelength may becentered at the 1234 cm-1 symmetric phosphate bond vibration absorptionpeak typical of the DNA backbone. The other QCL 3308 wavelength may beat a nearby reference wavelength measuring water absorption within thechannel. In this manner, water displacement by the cell may bereferenced out of the overall measurement. The mid-IR beam from thefirst QCL 3308 here is turned by a plain mirror 3310 and then combinedwith the beam of the second QCL 3308 by a dichroic filter 3312. A lens3314, which may be a reflective or refractive element, focuses theinfrared beams onto the measurement volume 3304 in the microfluidic chip3302. The transmitted infrared radiation (the input beam minus theabsorption of the sample in the channel) may be transmitted via a foldmirror 3322 to a mercury cadmium telluride (MCT) detector 3324 thatmeasures intensity.

The different wavelength QCLs are modulated/pulsed so their signals maybe separated in the electrical output of the MCT detector. Optionally,an additional dichroic filter and detector (not shown) may be used toseparately measure a particular IR wavelength. The detector may be a MCTdetector that is uncooled, TE-cooled, or even liquid nitrogen cooled toachieve maximum signal-to-noise ratio. Optionally, the detector may be athermal infrared detectors such as a pyroelectric or bolometricdetector.

In an embodiment, a reference detector placed on the laser side of thesample (not shown), may be used to measure laser output power in thecase where there are significant fluctuations in laser power. In thiscase, a small fraction of the beam may be sampled with apartially-reflective mirror, and a MCT or other detector used to measurepower before the sample and chip absorption. This signal may be thenused as a baseline for measurements.

As described earlier, multiple signal processing techniques may be usedto establish baselines in this system and accurately measure DNA contentin cells. Most of these may be well known in the fields of cytometry,vibrational spectroscopy systems and time-series infrared absorptionmeasurements.

A visible, NIR, or SWIR laser 3328 may be integrated into the system forthe purpose of detecting cell presence, and possibly size and density.The visible beam may be combined with the infrared beam using a dichroicfilter 3318, focused through the measurement volume, and then sent viaanother dichroic filter 3320 to a visible detector 3330. Additionaloptics, such as masks, may be added into the system ahead of thedetector 3330 in order to remove the unscattered light from themeasurement (achieved by blocking the zero-order light).

The microfluidic chip 3302 may be constructed in a method describedabove so as to maximally transmit infrared light, and reduce any opticalcavity effects. The infrared light may also be pre-treated by devices toreduce coherence length, and further minimize coherent optical effectsthat could introduce spatial dependence in the measurement channel. Onesuch device is described in more detail below.

FIG. 34 shows an example of the present invention using coherentanti-stokes Raman spectroscopy (CARS) to measure vibrational bondfingerprints in cells at high speed. The system consists of a pump laser3402 that may be combined with one or more slave lasers 3404 to exciteone or more bond vibrations in the cells being measured. The combinationof pump wavelength and specific slave wavelengths may serve toresonantly excite specific molecules so as to emit coherent light at ananti-Stokes frequency.

In the example shown in FIG. 34, the pump laser 3402 may be folded bymirror 3408 and combined with two slave wavelengths from lasers 3404using dichroic filters 3410. The pump may be typically pulsed in synchwith the slave (one slave at a time) to maximize signal. The pump andslave wavelengths may be focused by lenses onto the measurement volume3414 and any cells contained within it. The forward coherent Ramansignal (“F-CARS”) may be separated using a wavelength-selective filter3418 and focused onto a detection system 3420 that may typically consistof a photomultiplier tube (PMT) and associated electronics.

In addition, an epi-detected CARS (“E-CARS”) signal may be measured byseparating backward emission from the sample with a dichroic filter 3422and focusing it on a detection system 3424. Additional optical detectorsmay be used to detect the pump beam as a direct measurement ofscattering by the cells in the volume. In one example, pump beam may bepulsed alternately with each of the two slave wavelength lasers, eachcorresponding to a different vibration band or, one to a vibration bandcorresponding to DNA, and another to a reference band. Coherent Ramansignal generated by these pulses from the sample may be read out by thedetectors, and processed by the system to calculate cellular DNA asdescribed previously.

Although the laser wavelengths differ, and the readout mechanism may bedifferent, the fundamental “molecular bond fingerprint” being measuredcan be the same as when QCLs are used to interrogate the cells.Similarly, many of the same issues come to bear in the system. Forexample, good AR coatings on the microfluidic channels may be requiredto minimize variation as a result of mechanical or temperaturevariations, and to minimize the position-dependence of the laserintensity received by the cell (and the emitted signal intensity).

FIG. 35 shows a configuration of a QCL with components to reducecoherence length, so as to minimize spatial dependence in the readout ofcell spectral measurements. The QCL 3502 may be collimated by optics3504 and then passed through a diversity of phase shifts introduced byphase plate 3508. This plate may be spun or translated mechanically soas to provide rapid changes in phase to each part of the beam, with atime constant shorter than the integration time of the system.Additional optics 3510 then focus the beam onto the microfluidic chip3512 and the associated measurement volume, and the transmitted beam3514 is relayed to detector systems. The “decoherence” device may beapplied to multiple overlapping beams from multiple QCLs (at differentwavelengths) simultaneously. Such a device reduces coherence length, andreduces coherent effects near the focus of the system.

FIG. 36 shows an example of the display/user interface when the tool maybe used for cell sorting. The x-axis 3602 of the graph displayedindicates cellular DNA content. The y-axis 3604 indicates cumulativecell count. The histogram 3608 shown is generated from multiple cellularmeasurements. The tool may run a small sample of cells in order to buildup a distribution for display to the user or for fitting curves to thedistribution automatically.

In this example, a fitted distribution curve 3610 may be superimposedonto the histogram in order to indicate the spread of a particularpopulation of cells. For single-purpose tools, or for repeated protocolsperformed with a general-purpose tool, these fitted curves may begenerated and applied automatically. For example, in a tool based on thepresent invention meant to enrich sperm cells to either X-carrying orY-carrying populations, two such curves may be fit automatically ontothe data, and the user then simply selects an enrichment fraction andnumber of cells. A selection range 3612 indicates which range ofmeasured DNA content may be selected using the sorting function of thetool. This window may be manipulated directly by the user for someapplications.

Alternatively, the user may use the panel 3614 to simply specify thenumber of cells required, and the enrichment percentage desired. Thetool may then automatically calculate the optimal position for theselection window 3612 and calculate the estimated run time for the sort.

In an embodiment, where the present invention may be used for sortingsperm for gender selection purposes, the user interface may besimplified further: a) select X- or Y-enrichment, b) select desiredenrichment level, and c) select number of cells desired. The system thendisplays estimated run time, and if this is within an acceptable range,the user may initiate sorting. As the sort proceeds, the projected sorttime may be updated based on how many cells are being selected. Inaddition, the system may automatically alter the sort window 3612 inorder to achieve the targeted enrichment.

Other examples of single-purpose tools or protocols enabled by thepresent invention may include but are not limited to tools to separateout dividing cells, non-dividing cells or cells exhibiting aneuploidybased on the DNA content. Where double, or at least excess DNA canindicate cell division is in progress. Normal DNA content can indicatenon-dividing cells. Abnormal DNA content such as a low DNA content canindicate aneuploidy cells.

A system may include one or more of a liquid flow cell which isspecifically designed for high-speed measurements using a mid-IR QCL, anelement for creating phase diversity in the mid-IR laser light, anoptical subsystem that ensures a narrow, focused spot is sampled in theliquid sample, an element minimizing the scattered or reflected lightthat reaches the detector, and a QCL with a rapid, low-cost, coarsetuning mechanism specifically suited to liquid- or solid-phasemeasurements. Rather than making a surface measurement of a liquid suchas that performed by existing mid-IR sampling architectures, this systemmakes a transmission measurement through the entire sample, in whichmid-IR light is transmitted directly through the sample. The systemminimizes reflections of any type, and methods may be used with thesystem to reduce contributions from scattered or reflected light.

Depending on the speed at which the absorption spectrum of the liquidmust be measured, and the QCL power available, the path through the flowchannel must likely be designed to be short because mid-IR absorption ofliquids (water, in particular) is often very high. Typically, a pathlength on the order of microns is required—for very high-speedmeasurements, 25 microns or less. A limiting factor, even in the casewhere laser power is not, is the total power absorbed by the liquid, andany temperature constraints inherent to the cells or the liquid (forexample, it could complicate matters significantly if the liquid startsboiling in the channel).

Such a flow channel may be fabricated in one of a number of ways knownto those skilled in the art. For example, multiple devices with flowchannels can be fabricated by patterning at least one of pairs of waferswith multiple adjacent flow channels either by etching into the wafer,or by adding and then patterning another material on top of the wafer.For example, SU-8, a photopatternable polymer, may be used to patternflow channel walls on one of the wafers. Wherein the wafers aretransparent to mid-infrared light. The wafers are then aligned to eachother and the wafers are bonded together with an adhesive to provide alaminated mother wafer. SU-8 can also be used as an adhesive forexample. Finally, these laminated mother wafers are diced intoindividual devices. Wafers may additionally be patterned and etched toform inlets and outlets for liquids. Channels may be narrow, or may infact be large areas.

In some cases, the “channel” may in fact be a large liquid volumebetween two windows, and rather than the liquid flowing past thedetection point, the sample holder may be translated past a measurementsystem—with the sample holder (windows and interposing liquid space)designed as described below.

For the purpose of consistent measurements of the liquid and anycontents therein and improved accuracy of the measurements, it isimportant to take into account coherent and resonant optical effectsthat may occur in such a measurement system. Such effects may include:laser (QCL) coherence effects which cause a varied field strength andtherefore absorption over the sample volume, causing position dependencewithin the measurement, either along the axis of the laser (depthdependence) or laterally (x-y dependence or “speckle”). Reflective orsemi-reflective surfaces adjacent to the sample, which can result in aspatial variation in EM field strength near the surface (physically, amirror will have a field minimum at its surface); and reflective orsemi-reflective surfaces which interact to form a resonant cavity,resulting in: wavelength-dependence in absorption due only to resonanceor lack or resonance (effectively, some wavelengths will have moreaverage passes through the liquid sample than others); and again, atfixed wavelength, result in a static distribution of field strengthwithin the sample, making it unevenly sampled and position-dependent.

The present disclosure includes a number of specific design parametersfor the liquid channel/sample holder and the optical delivery system tominimize these issues and assure higher accuracy in absolutemeasurements of the sample. These may be applied singly, or incombination, to minimize position-dependent absorption within the flowchannel of the mid-IR beam(s).

One specific design parameter may be fabricating the sample holder withwell-designed antireflection (AR) coatings on both external (air-facing)and internal (liquid-facing) surfaces. These coatings preventreflections at the interfaces, which is important both for eliminatinglocal field minima/maxima near the interfaces (which may causenon-uniform “sampling” of the liquid), and reducing resonant opticaleffects within the system. AR coatings for glass or plastic to airinterfaces are well known for mid-IR wavelengths. If the index of theliquid sample is close to that of air, these may be sufficient forinternal (liquid-facing) surfaces as well. However, the AR coatings mayrequire special design, including: matching of the AR coating tominimize reflection between the window (which is a material transparentto mid-IR radiation, for example Zinc Selenide, Silicon, Germanium,Barium Fluoride, Calcium Fluoride, and certain plastics with high IRtransmission) and the liquid analyte (water, for example); designing theAR coating for the specific wavelength, or range of wavelengths, to beused in the spectral analysis system; designing the AR coating for the(range of) angles of incidence of light that will be seen by thesurface, wherein the angle is a combination of the beam cone angle (mostsystems will be focused down to the channel) and the angle at which thesample holder is placed relative to the beam (see discussion below);designing the AR coating for compatibility with the liquid analyte (manymid-IR coatings are comprised of materials that absorb water so thateither alternate materials must be used, or capping layers that resistwater penetration must be used); and/or terminating the AR coating withlayers, or post-treating the AR coatings in order to make the surfacewith the appropriate hydrophilic properties required to move fluid intothe channel or cavity; possibly coating or treating the surface in orderto decrease (or, in rare cases, increase) adherence of biological orother particles to the surface; for example, in a flow cytometryapplication, ensuring that cells do not adhere to the channel walls inthe measurement volume (or elsewhere); these terminal layers ortreatments must of course not significantly reduce the effectiveness ofthe AR coating itself.

Another specific design parameter may be angling the surfaces of thesample holder such that any reflections are rejected from the system andnot delivered to the detector, or reflected back to the laser source.The cone angle of the light focused onto the sample holder should betaken into account in this case. In addition, AR coating designs may bemodified to take into account this angle of incidence. Angling theentire channel may effectively increase the path length through theanalyte. This may be beneficial in some cases but needs to beconstrained in others where the liquid analyte (or liquid carryingparticles of interest) is highly absorptive at the target wavelength(s).The beam may be asymmetric in order to achieve higher uniformity inmeasurements. For example, in a flow application it may be desirable tohave a short axis parallel to the flow, and long axis across it in orderto have the most uniform illumination across the center of the flow. Toprovide the most uniform illumination, the cone angles provided by thelight at the center of the flow should be taken into account. In thiscase it would usually be desirable to tilt around the short axis, sincethe cone angle along the long axis will be narrower, and therefore lesstilt will be required to ensure no reflected light is sent to thedetector or laser subsystem.

Another specific design parameter may be using nonparallel surfaces inthe measurement volume in order to minimize resonant effects. However,the optical path through the liquid should be consistent in cases whereparticles in the liquid are to be measured, so that the attenuation dueto liquid absorption is identical regardless of particle position withinthe sample volume.

Another specific design parameter may be employing specific gapdistances through the channel, tuned to the interrogating wavelength(s)and the average index of the liquid analyte or liquid carrier plussample particles. A channel thickness (or gap) may be specificallynon-resonant at the wavelength of interest, meaning that the gap timesthe index of the liquid contained in it are a non-quarter wave multipleof the interrogating wavelength. This prevents constructive ordestructive interference effects to the maximum extent possible,reducing buildup of resonances in the cavity, and in this mannerminimizing spatially dependent absorption in the system.

The system is focused on enabling high-speed liquid-phase (or solidparticles/cells within a liquid medium) spectral measurements. Liquid-and solid-phase absorption lines in the mid-IR are far broader (severalwavenumbers at a minimum) than gas-phase absorption features. As aresult, it is acceptable to have broad linewidth, but at the same time,it is necessary, to effectively have a wide tuning range in order toreach one or more absorption peaks of interest, as well as referencepoints in the spectrum which are used to establish a baseline. For thesake of speed, this tuning must be performed very rapidly over thisbroad range. So the requirements on the QCL subsystem are very differentthan for gas-phase spectroscopy. The system is described as separatedelements in a system, as some applications will benefit from thesimplicity of having a separate microfluidic element—that may bedisposed of after use in some applications, or at least swapped outperiodically.

One of several designs for the QCL subsystem may be used in the presentdisclosure. One design is discrete fixed-wavelength QCLs that areoptically multiplexed. In this design, individually packaged QCLs areused. They may be either distributed feedback (DFB) lasers or externalcavity Fabry-Perot (FP) type lasers (where wavelength is set in theexternal cavity). The outputs of the lasers are collimated, and arecombined with one another, typically using either dichroic or bandpassfilters (which, for example, reflect one wavelength and transmitothers). If power is not an issue, semi-transmissive mirrors may be usedto multiplex the beams. These lasers may then be pulsed in rapidsuccession to measure absorption at different peak and referencewavelengths of the liquid in the microchannel. Alternatively, if theQCLs are operated in continuous wave (CW) mode, they may be modulatedwith different frequencies and their signals demultiplexedelectronically after detection by a mid-IR detector (after passingthrough the liquid sample). Optical demultiplexing to multiple detectorsis also a possibility, where the highest signal-to-noise ratio isabsolutely necessary. The advantage of multiple discrete QCLs in thepresent invention is that they are more readily available from supplierstoday, and that they may be changed relatively easily (for example, ifsystems with different chemical targets are being built). In addition,they may span very broad wavelength ranges, whereas the tunablesolutions described hereafter will cover a relatively narrow range ofwavelengths (and therefore may require the use of several tunable QCLsubsystems within the present invention).

Another design is the QCL array-on-chip. This design consists of anarray of DFB lasers fabricated on a single chip. This means a singlegrowth design is used, but gratings that set laser wavelength areindividually patterned to result in different center wavelengths. Thepotential advantage of this architecture is the lower cost of packaging,cooling elements, and associated elements. It also opens the potentialfor a very compact system that may be rapidly switched from onewavelength to another simply by electronically switching between lasersin the array. The array may be produced with regular-spaced wavelengthover the interval of interest (which covers one or more absorption peaksin the liquid sample, and perhaps one or more reference absorptionmeasurements). Wavelengths may be spaced apart at wider intervals, andeven at irregular intervals. For example, the array could contain 4discrete and unevenly-space wavelengths over a frequency interval of 200cm-1, corresponding to peaks and references. For example, if cellularDNA in live cells flowing through the liquid channel is to bequantified, and therefore the characteristic symmetric phosphate bondpeak at 1087 cm-1 is to be measured (phosphate is an element of the DNAbackbone), three closely-spaced wavelengths at 1075 cm-1, 1087 cm-1 and1099 cm-1 could be employed to measure absorption peak absolute heightand “shape,” and another wavelength at 1055 cm-1, corresponding to anearby absorption minimum (and therefore potentially good referencelevel) could be added. In this manner, a single chip containing allrelevant wavelengths may be fabricated and packaged with minimum sizeand cost. The beams from the lasers on the common chip may be deliveredin at least two ways to the microfluidic channel: 1) A grating may beused to redirect individual wavelengths in a manner such that a single,overlapping collimated beam is formed. The coincident beams may then befocused onto the microfluidic channel where they are absorbed by theanalyte, and then relayed to a detector. Note that for irregularlyspaced wavelengths, laser diodes may have to be spaced accordingly onthe chip in order to have a diffraction grating accurately redirect thebeams into a single overlapping beam. Or 2) The plurality of lasers maybe imaged directly onto the microfluidic channel using appropriateoptics. In this case, an array of points corresponding to the array oflasers will be projected onto the microfluidic channel. In a 1:1 imagingsetup, for example, if the DFBs are patterned 20 microns apart on thechip, a series of spots 20 microns apart will be sampled in the liquidchannel. For example, these could be oriented along the direction offlow of a micro-channel, and the liquid and anything being carried bythe liquid would be sampled sequentially by the beams emanating from theseries of lasers on the chip. The wavelengths of the lasers could inthis case be in any pattern; for example they could simply be analternating array of two wavelengths (signal and reference wavelengths).In other configurations a larger number of wavelengths could be used tobuild up a rough spectrum. If configured in this way, in a system whereparticles such as cells are run through the liquid channel, the laserscould in theory be run in CW mode, if appropriately spaced, and signalprocessing could be used to extract the absorption levels. In mostcases, however, individual lasers will still be pulsed in rapidsuccession, or modulated at different frequencies to provide for easyelectronic separation after detection by a mid-IR detector. The spotsfrom the array may be imaged such that they are not entirely parallel tothe microfluidic channel. They could be oriented in a diagonal manner inorder to give some lateral resolution to the system. Such lateralresolution within the flow channel could be used for example to measureposition of cells within the channel as they flow by (to compensate forany known/calibrated spatially-dependent detection nonuniformities), or,for example, to measure concentrations across a flow withnon-uniformities, from either differential velocities (center vs. edge),or because two liquids are mixed upstream in a largely laminar flow.

Another design may be a QCL that rapidly tunes to discrete wavelengths.As discussed earlier, continuous or fine-stepped tuning is not requiredfor the liquid spectroscopy application. High speed however, is arequirement. A rapid tuning mechanism that samples sparse wavelengthswould be ideal. One tuning mechanism that lends itself to potentiallyrapid, discrete, controllable, low-cost tuning is a Vernier tuningarrangement in the external cavity of a FP QCL. In such an architecture,thermal tuning (for example) can be used to achieve much faster, broadertuning than would otherwise be possible. Such architectures are wellknown and have been used to build telecommunications lasers in the 1.5micron range. We propose that such an external cavity architecture for amid-IR (or eventually THz) QCL, integrated into a system with a fluidmicrochannel in which absorption is measured, would be a potentiallyideal solution for high-speed liquid spectroscopy. In the Vernier filterconfiguration, two FP resonant elements are used in the external cavity,designed with free spectral ranges (FSR) that are close but not equal.By slightly tuning one or both of these elements, different transmissionpeaks will coincide. In this manner, the portion of the gain spectrum ofthe QCL that is amplified is selected. The advantage is that withrelatively little input signal to the FP cavities (in the form oftemperature in thermo-optically tuned systems, or voltage inelectrostatically-tuned systems), large hops in wavelength may beachieved. Furthermore, the wavelength settings themselves are relativelywell controlled since the modes of the FP cavities are known. Thespacing and centers of these wavelengths may in fact be optimized withinthe present invention to provide the most efficient measurement of theliquid or liquid-suspended analyte. For example, the spacing of emissionpeaks could be configured to fall on the absorption peak of interest andon a reference point only for maximum efficiency. With the addition of athird filter to the external cavity of the QCL, one could sample a fewwavelengths around the absorption peak, then a few wavelengths at one ortwo reference points.

More conventional tunable QCLs (such as those tuned by piezo-actuatedexternal gratings) may also be used in the context of the presentinvention, in conjunction with other elements described herein, so longas they provide the required tuning speed.

Unlike with hot filament blackbody (“glowbar”) sources used in FTIRinstruments, QCLs are inherently coherent optical devices, and a numberof potential complexities arise as a result. Coherent effects in imagingsystems are well known (“laser speckle”) and arise from constructive ordestructive interference of the laser light returning from variouspoints in the sample. We wish to avoid such effects, to the extentpossible. A few elements described below may be employed to ensure thatcoherent effects—particularly spatially-dependent effects—are minimized.One element involves optical apertures—optical apertures through whichthe interrogating beam is focused may serve to reduce optical effectsfrom the laser, and provide higher consistency in measurements. Anaperture on the QCL side of the system (before the sample) may be usedto “clean up” the beam from the QCL, and therefore deliver the minimumspot size onto the sample in the microfluidic channel or cavity. In thisconfiguration, lenses are used at the output of the QCL to focus mid-IRlight through an aperture of roughly 20 microns or less (approximatelythe desired spot size on the sample). This aperture will be imaged ontothe sample, and ensure that a “clean,” small spot is sampling theliquid. An aperture on the detector side of the system (after the beampasses through the sample) may be used again to “clean up” the beam—thistime also removing any scattered light from the measurement.Combinations of scattered and directly transmitted light in a coherentsystem could cause unwanted effects. In addition, elimination ofscattered light from the measurement ensures that only light that haspassed more or less directly though the sample (and therefore reflectiveof Beers law absorption) will be measured in the system.

Another element involves phase scramblers. Phase scramblers may be usedon the input (QCL) side of the system in order to de-cohere the lightimpinging on the sample. Scramblers typically rapidly change opticalphase across a beam, on a time scale some multiple shorter than themeasurement time (for a single event). In this manner, coherent effectssuch as those described above are effectively “averaged out” over anumber of states. One example of a phase scrambler is a transmissivedisc that has been etched with a pseudo-random pattern of fields withdiffering phase delays (by virtue of material index and thicknessdifferences) as described presented in FIG. 35 and described previously.This disc is the spun at an angular rate sufficient to “scramble” phasesover a single measurement, and the beam from the laser is sent throughthe disc before it is focused onto the sample. We propose such a phasescrambler, designed for the mid-IR, combined with a mid-IR QCL, formicroscopic spectroscopy applications in the mid-IR such as the onesdescribed herein.

The present disclosure may be applied to, and form the core of, multipletypes of systems that rely on rapid liquid-phase spectral measurements.

One such system is a monitoring system for liquid phase reactions. Insuch systems, very small volumes of reagent are used in a microchannelor microcavity, and the ensuing reaction is measured. The advantage ofthe present system is its capability to measure chemical concentrations,or even changes in chemical configuration (shape, folding, etc.) at highspeed in liquid phase, and producing accurate results on an absolutescale (rather than just a time series of relative measurements). Suchcapability could be used for medical diagnostics, environmental tests,or combinatorial chemistry done at large scale on a chip.

Another such system is a high-throughput screening system. Manytechnologies have been devised to measure changes in cellular behaviorin response to potential drugs. Most of these sample the cellsuperficially (for example, with surface-oriented optical techniques),and/or do not have the ability to directly measure key biochemicalconcentrations within the cell. The present invention enables suchmeasurements to be made, consistently, in the native liquid environment,and at high speed. High speed may enable either very rapid successivemeasurements in a single cell, or will allow the system to rapidlymeasure a large number of cells that have placed in reaction wells withcompounds. The ability to sample small volumes of liquid, containingcells, in a manner not dependent on cell position within the volume—andto do so accurately and quickly—is an advantage of the presentdisclosure.

Another such system is for high-speed cell classification. Emergingtechnologies that trap cells in a microfluidic cavity using structuredand/or coated surfaces often require follow-up measurement and screeningof cells to classify them. Often extremely rare cell events are ofinterest. For example, circulating tumor cells, or embryonic cellswithin the mother's bloodstream are typically 1 in a billion or less infrequency. Microfluidic structures may serve to enrich these to 1 in1,000 or 1 in 10,000 in frequency. Subsequently, the trapped cells(which include many white blood cells, for example), must be screened inorder to find true occurrences of the rare cells. The cells identifiedmay then be extracted for further analysis. The present invention may beused to build a system for performing these screens rapidly on apopulation of cells that are trapped within a microfluidic cavity. Knownbiochemical markers established with FTIR or Raman spectroscopy inresearch studies may be interrogated using the present invention toaccurately and rapidly screen and classify cells. Stem cell cultures maybe measured in a similar manner to determine state of differentiation,while in a microfluidic cavity.

Another such system may be rapid cell counting/classification in a flowsystem. The present disclosure may be embodied in a format where asuspension of cells, or bodily fluid containing cells, flows through amicrofluidic channel, and cells are interrogated as they pass throughthe mid-IR beam. The absorption of one or more wavelengths is measuredby the system, and the cell is classified, or measurements are directlydisplayed to the user as a histogram. For example, cellular DNAmeasurement for cell cycle characterization may be done. Distribution ofDNA quantity in a cell sample is indicative of how quickly cells in thesample are dividing and therefore multiplying. Cells with twice the“normal” DNA are in the process of dividing. In another example,cellular DNA measurement for aneuploidy detection may be done. Unusuallevels of DNA are often an indicator for cancer. Detection of an unusualdistribution of DNA within a sample can be a strong indicator that asample is cancerous. In another example, blood cell counts may bemeasured. A complete blood count, including counts of all white bloodcell types, may be possible using embodiments of the present disclosure,without the use of fluorescent labeling or other preparation. Cellswould be classified using a combination of mid-IR wavelengths,potentially in combination with visible-light detection schemes. Besidesindividual cell measurements and counts, the liquid content of the bloodplasma itself could be analyzed using the same system. In anotherexample, an analysis of semen could be performed using embodiments ofthe present disclosure. Sperm cells could be counted and potentiallycharacterized (for proper DNA packaging and chromosome count, gender);other cell counts could be determined (white blood cells); seminal fluidcharacteristics measured.

Another such system may be a cell sorting system. Well-known sortingmechanisms may be added to the cell measurement systems above in orderto sort cells into distinct populations based on the measurementsdescribed. For example, sperm cells may be sorted for gender, cellsexhibiting abnormal DNA count (potential marker for cancer) may beselected for analysis, cells that are actively dividing may be selectedto create a sample with high viability, and cells may be sorted based onother biochemical markers; for instance in development of biofuels,cells that are determined to be producing a large amount of the targetchemical may be identified spectroscopically without destroying thecell, and cycled back into the cell culture, while “less successful”cells are disposed or sorted to a different collection point.

Another such system may be for embryo scoring. In in-vitro fertilizationprocedures, it is strongly desirable to implant the minimum number ofembryos required for a successful pregnancy. To this end, there issignificant research ongoing into “scoring” embryos for viability, basedon biochemical and/or morphological changes in early development. Thereis research that indicates biochemical concentrations could be a keyindicator for viability. Embodiments of the present disclosure could beused to interrogate and score embryos, in a liquid environment, withoutthe use of dyes or labels, and using only very low photon energy lightto enable the embryo to be interrogated and scored without damaging theembryo. The ability to rapidly measure spectra, and potentially trackspectra over early development, could be a significant advance in theability to score and select embryos for implantation.

Another such system includes a gas monitoring system. In some cases,monitoring gas flows directly (sometimes done using mid-IR spectroscopy)is not practical. In such cases, it may be possible to flow the gas overor through a liquid stream that reacts with the target compound in thegas. The liquid stream then is flowed through a microfluidic channelwhere it can be interrogated using the present invention. We proposethis system as a whole as an effective, compact, rapid manner ofmonitoring some gas compositions—for example, detecting trace impuritiesin gases, or detecting biological or chemical agents in air. Similarly,a liquid surface may be exposed to air or another gas flow for aspecified duration, and then the liquid, together with any particlestrapped over that duration, analyzed using the present invention.

Another such system may be a solid sampling system. Sampling of solidsby infrared spectroscopy is a long-standing challenge. There is verylittle optical penetration into most solids by mid-IR. As a result,surface techniques such as attenuated total reflectance (ATR) must beused, which are often limited by surface layers or texture. The liquidspectroscopy system described herein may be used in an embodiment, wherea solid is sampled by mechanically fracturing/grinding it into verysmall particles, filtering these particles to ensure a reasonable rangeof diameters/shapes, and then suspending them in liquid for subsequentmeasurement. Measurement can then be performed in a cavity where thesolid particles are dispersed across an area (or a line), or with liquidthat flows through a channel through a small measurement volume.According to the designs laid out above, the liquid and suspended solidsare then measured using mid-IR transmission. In one embodiment, onemid-IR wavelength may be used to measure water absorption, in a rangewhere the solid of interest does not absorb strongly in the mid-IR. As aparticle moves through the optical detection area, its volume may beestimated by the decrease in water-line absorption. At the same time (orin rapid succession), the absorption of the solid particle at the targetwavelength is measured. It may then be normalized for particle volume inorder to calculate the chemical composition of the particle. An exampleembodiment is an environmental sampling tool. A tool with a small drillis constructed to drill into a layer suspected of being asbestos. Asmall amount of liquid is injected, and then a capillary-type tube isused to sample this liquid with any suspended solid. This liquid is theninterrogated at the appropriate wavelengths to determined chemicalcomposition and structure to signal whether the substance is asbestos.Pharmaceutical purity inspection is another example. Another such systemmay be an emulsion measurement system (oil in water; water in oil).

FIG. 37 shows a sample embodiment of the present invention that includeselements to reduce spot size on the sample, and to reject scatteredlight from the sample. A QCL subsystem 3702 which may consist of one ormore mixed or tunable QCLs in the mid-IR or THz range may be collimatedand then refocused by lenses 3704 and 3708 into an aperture 3710 thatserves to “clean” the beam from the QCL and ensure a smaller-size,Gaussian beam profile at the sample. Lenses 3712 and 3714 then refocusthe beam with a yet smaller spot size onto the fluid microchannel in thesample holder 3718. The sample holder 3718 is shown here at an angle toensure that stray reflections are not propagated to the detector, and toreduce any resonant optical effects, but other geometries are alsopossible. The flow of the liquid shown here by arrows may be a samplethat may be transported through a channel, through the measurementvolume and microchannel that the QCL-beam is focused onto. Lenses 3720and 3722 collect the light that transmitted through the liquid in thesample holder 3718, and refocus it onto another aperture 3724 which canblock scattered light from the sample or sample holder, and therebyrestrict light reaching the detector 3732 to directly-transmittedradiation. Lenses 3728 and 3730 deliver the light to a detector 3732,for example a mercury cadmium telluride (MCT) detector for the mid-IR(which may be cooled by thermoelectric elements or liquid nitrogen). Byblocking scattered light and restricting the light that reaches thedetector to only directly transmitted light, the disclosed methodprovides an improved absorption measurement of liquid samples withsuspended particles/cells.

FIGS. 38a-c show several sample configurations well known in theindustry for performing mid-infrared measurements that may suffer fromsurface effects that the present invention is specifically designed toavoid. FIG. 38a shows an ATR configuration often used to measure liquidsor solids using Fourier Transform Infrared (FTIR) spectroscopy in themid-IR. Here it is shown in contact with a liquid micro channel showingflow velocity in the cross-section. As is usually the case in suchsystems, velocity near the interface may be very low. Also shown is thelimited penetration of the evanescent field from the ATR prism into theliquid flow. The advantage of using ATR in conventional FTIR systems isthat even when liquid absorption is very high, very little light may beabsorbed in this configuration. The strong disadvantage obvious fromthis figure may be the limited depth of penetration into the core of theflow. FIG. 38b shows a more recent configuration used by a number ofgroups, which may be similar but uses plasmonic layers (patterned metalconductive layers) to enhance absorption. This may strongly enhanceabsorption signatures of samples in direct contact with the plasmonicfilter. Again, however, the mid-IR field may have very limitedpenetration into the liquid. For measurements of stationary cellsadhered to the substrate as shown by one of the biological cells in thisexample, this may allow time series measurements. However, forhigh-speed interrogation of cells passing through a liquid microchannel,this may not be an appropriate architecture, because of the strong depthdependence and limited depth of the absorption. FIG. 38c shows a truetransmission architecture that has been used for mid-IR measurements byseveral groups. In this “transflection” architecture, mid-IR lightpasses through the sample, is reflected by a mid-IR reflective substrate(which may be transmissive in the visible), and then makes a second passthrough the sample before proceeding to the detector. Measurements havealso been performed with an “open channel” architecture, where there isno top window on the flow and the liquid sample flows for a limiteddistance over the reflective substrate. The advantage of this may beimproved transmission and more simple construction. The strongdisadvantage is that any variability in liquid layer thickness in theopen channel flow may result in large apparent changes in sampleabsorption. A more substantial problem with the transflectionarchitecture, however, derives from interference effects resulting fromthe reflective substrate. For example, a cell very close to thereflective surface, where the electric field must necessarily fall to alow level, may absorb relatively little mid-IR light. Conversely, a cellat a certain distance from the substrate will absorb a larger amount oflight. This variation in signal depending on the depth position of thecell in the flow is difficult to compensate for in interpretingmeasurement data. In addition, in this architecture it may be difficultto distinguish which light is reflected by the sample, scattered by thesample, or transmitted through the sample. The present invention seeksto remove most of the issues by providing new methods and apparatus.

FIG. 39 shows an example embodiment of a microfluidic channel describedin the present invention. A fluidic channel 3902 may carry fluid and, inthis example, biological cells 3904. The channel may be fabricatedbetween two mid-IR transparent windows 3908, whose thickness is notshown to scale (typically, the channel will have a thickness on theorder of 10's of microns, and the windows will have thickness on theorder of 100's of microns). The windows may be fabricated from mid-IRcompatible materials such as Germanium, Silicon, ZnSe, CaF2, and thelike. The windows may be antireflection coated, with a coating toprevent reflection at the air interface 3910, and another coating on theinternal surface 3912 tuned to prevent reflection at the liquidinterface. A mid-IR beam carrying one or more mid-IR wavelengths 3918may be then focused onto a sample volume in the fluidic channel.Indicated here is the average path length 3914 which may also be tunedto minimize any resonant effects in the fluidic cavity. In this example,the beam may be brought in at an angle to guide any stray reflectionsaway from the detector, and to minimize any resonant effects in thefluidic channel. As cells pass through the measurement volume,absorption changes at one or more mid-IR wavelengths which forms thebasis of the measurement of the invention. The system may detect thesignals corresponding to the absorption levels, remove backgroundlevels, and calculate the chemical concentration of one or more cellularconstituents. Measurements at different wavelengths can be performedsimultaneously or serially. For example, DNA levels may be interrogatedusing the present system. The microfluidic channel may be fabricated ona disposable microfluidic chip and carrier so as to preventcontamination. An alternative configuration does not use a flowingchannel, but rather a 2-dimensional planar cavity in which many cellsare immobilized. The chip may then be translated in x- and y-directions,possibly guided by a visible-light system that identifies candidate celllocations, and mid-IR is used to interrogate cells. For example, incirculating tumor cell (CTC) applications, a 2D microfluidic pattern canbe used to trap rare CTCs in blood. The present invention may be used toscan the trapped cells, and detect actual CTCs among white blood cellsand other particles trapped in the array.

FIGS. 40a-c show how a microfluidic cavity may be designed to reduceresonant optical effects, such that as much position dependence aspossible is taken out of the QCL-based fluidic measurement system. FIGS.40a and 40b show a microfluidic gap which may be an even quarter wavemultiple of the interrogating wavelength, which is a peak resonancecondition. FIG. 40a shows the electric field at resonant conditions inthe microfluidic gap, while FIG. 40b shows the resulting opticalintensity in the microfluidic gap. As may be seen, there can be a strongpeak in the electric field and optical intensity near the center of themicrofluidic gap. In general, it is desirable to avoid such strongspatial dependence to provide a more uniform measurement conditionacross the microfluidic gap, because particles may be positioned atvarious heights within the microfluidic gap. FIG. 40c shows a preferredconfiguration, where a gap is provided that is not a quarter wavemultiple and as a result is not resonant at the interrogatingwavelength(s). This non-resonant design of the microfluidic gap, andconsequently provides more uniform measurement conditions across themicrofluidic gap. This configuration is further improved by including ARcoatings and angling as described previously.

FIG. 41 shows an embodiment of the present invention based on aVernier-tuned external cavity QCL. This type of laser, which may be awell known architecture, can be ideal for certain liquid spectroscopyapplications, because these applications require only rough but fasttuning to a relatively small number of emission peaks within aparticular wavelength range. Several of these lasers may be used tointerrogate a liquid and/or suspended solids within a particular system.In this system, a gain medium 4102 emits mid-IR light on its rear(low-reflectivity) facet (the left side as shown), which is collimatedby lens 4104, and then passes through two etalons 4108 and 4110. Thefree spectral ranges of these etalons may be slightly different, so thatonly one set of transmission peaks coincides over the gain range of thegain medium 4102. The etalon wavelengths may be tuned by thermal ormechanical means. Importantly, only slight tuning of the etalons may berequired to tune the wavelengths rapidly over a wide range. The abilityto provide large wavelength tuning steps is particularly compatible withliquid measurements where absorption peaks may be broad. A rear mirror4112 returns the beam back through the etalons 4110 and 4108 to the gainmedium 4102. Light emitted through the high-reflective front facet(right side as shown) of the gain medium 4102 is collimated andrefocused by lenses 4114 and 4118. Note a subsystem for “cleanup” of thebeam employing additional lenses and a small aperture may be used toreduce the spot size. The beam is then focused onto the liquid sampleholder 4120 including a microchannel or microcavity. The transmittedlight is then focused onto a mid-IR detector 4124 using lens(es) 4122.

FIG. 42, taken from U.S. Pat. No. 6,853,654, further illustrates theVernier tuning mechanism proposed for high-speed mid-IR liquidspectroscopy as part of the present invention. It shows the transmissionspectra of two etalons used to tune the laser, their different freespectral ranges (FSRs), and how there is a point where they coincide.

FIGS. 43a-b show examples of multiple wavelengths used in the presentinvention. The individual wavelengths may be produced by a tunable QCL(for example, the Vernier configuration specifically described herein),individually-packaged fixed-wavelength QCLs, or a monolithic QCL arrayon a chip, delivered either as individual beams, or combined into asingle spot. For the purposes of this figure, the horizontal axisrepresents mid-IR frequency, and vertical axis represents absorbance.FIG. 43a shows a configuration where an absorption spectrum is measuredat an absorption peak of interest 4302, with three points being measuredon the peak. This allows the shape derivative, or second derivative tobe measured as well as absolute absorption. Where measuring the secondderivative can be useful for determining an absorption peak in thepresence of broad background signals. In addition, a local absorptionminima 4304 may be interrogated by one of the system wavelengths. Thismay allow a measurement of the background absorption level, forinstance, of the liquid medium delivering cells or particulates into themeasurement volume. FIG. 43b shows a more minimal configuration, whereinonly a peak absorption wavelength 4308 and a reference wavelength 4310may be sampled.

FIG. 44 shows another example embodiment of the present invention. Asingle-chip QCL array 4402 may contain multiple QCLs at multiplewavelengths providing multiple laser beams. These may be collimated bylens 4404 and then treated using a rapidly-moving phase delay element4408 in order to reduce coherence in the system, as described earlier.Another lens 4410 can then be used to focus the beams onto themicrofluidic channel 4412. In this example, the laser array may beimaged onto the microfluidic channel 4412 such that a series of volumesare illuminated along the axis of flow in the microfluidic channel 4412.The transmitted portions of the beams may be then delivered to a mid-IRdetector 4418 via one or more lenses 4414. In this configuration,different points along the channel may be sampled by each laser and it'sassociated wavelength. For example, in a cytometry system for measuringbiological cells, a cell will sequentially pass through one beam afterthe next, causing different signals on the mid-IR detector. The changesin signal as the cell passes may then be processed, and chemicalconcentrations calculated. Simultaneously, the individual lasers may bepulsed sequentially such that individual signals based on the light fromeach laser are easily resolved. The lasers may be modulated withdifferent frequencies or they may be used in continuous mode. Thelocation of the cell can be inferred from the pattern of the detectedsignals as it moves through the microfluidic channel 4412. In a systemusing CW lasers, analog or digital differentiation may be used toisolate signals corresponding to absorption by a cell moving through themeasurement volume. The potential advantage of CW lasers, besides totaloptical power, is stability. CW lasers may be sufficiently stable tomake a series of fast, referenced measurements as one cell passesthrough the measurement volume with reference power levels read outbefore and after the cell is in the volume whereas pulsed lasers mayvary from pulse to pulse and require an additional detector in thesystem to reference QCL power.

One of the problems raised in mid-IR microspectroscopy is that ofscattering. Mie scattering is dominant when particles are on the orderof the interrogating wavelength. The magnitude and angle of scatteringis determined by the size of particles and the index of the particlesrelative to the fluid medium. Described is a high speed particlemeasurement system for measuring the chemical composition/content ofparticles in the mid-IR using QCLs emitting at a few discretewavelengths and optical system architectures to reduce or harnessscattering effects for the purpose of making these measurements.

Scattering is generally quite large in the visible regime, as the probewavelengths used are small relative to the cellular dimensions. Inaddition, measurements made in this regime are generally dependent onwavelength only insofar as scattering is dependent on wavelengths vs.feature size and refractive indices of many materials are relativelyconstant over wide ranges within the visible.

In the mid-IR, refractive indices of cellular components can vary quiterapidly as the molecular bonds vibrate at frequencies corresponding tothe mid-IR wavelengths. These molecular bond vibrations also cause themolecules to absorb light, resulting in absorption bands. In gases,these absorption bands are generally extremely narrow. In liquid andsolid substances, the absorption bands are broader. Because there arelocalized absorption bands, corresponding to raised imaginary componentsin the complex refractive index, there is necessarily a localfluctuation in the real refractive index of the compound, as may bedemonstrated or calculated from the Kramers-Kronig relation between thereal and imaginary components of refractive index as is well known tothose skilled in the art.

As a result, when designing systems for measuring particle spectroscopicproperties in the mid-IR, local refractive index fluctuations shouldalso be considered, and their effect on scattering as a function ofwavelength. In some cases, system design can be optimized to reduce theeffects of this scattering. In other cases, reference measurements canbe used to characterize the scattering intensity and compensate for itin an absorption measurement. Finally, in certain instances, scatteringand its strong wavelength dependence near absorption peaks may beharnessed to perform a measurement.

FIGS. 45a-b show a schematic example of a molecular absorption peak inthe mid-IR. FIG. 45a shows the absorption of the particle (α_(Particle))and the absorption of the medium (α_(medium)) in which it is measured,both as a function of wavenumber (ω) is commonly used in spectroscopyand ω is an inverse function of wavelength (ω≈f(1/λ)). FIG. 45b showsthe derived real refractive index of the particle (n_(PARTICLE)) and themedium (n_(medium)). As shown in FIG. 45b , there is a fluctuation inparticle refractive index around the center of the absorption peak. Theparticle has a baseline refractive index n_(PARTICLE) ∞ (“index atinfinity”) that may be different than that of the medium. For certainapplications it may be desirable to change the medium to have arefractive index closer (less scatter) or further (more scatter) fromthe particle. If a straight absorption measurement is desired, it isdesirable to reduce the index difference between the particle and themedium by using a liquid medium with a higher index, so as to minimizethe effect of scatter. On the other hand, if a scattering-basedmeasurement of particle size, for example, is desired, then it may behelpful to increase the index difference between the particle and themedium by using a liquid medium with a lower index relative to theparticle index.

FIG. 45b also indicates three wavenumbers associated with the particleindex, the center wavenumber for the absorption band ω0, a high indexpoint ω−, and low index point ω+. Maximum and minimum scattering mayoccur roughly around these points. In building a discrete-wavelengthmeasurement system for particles or cells, it may be crucial to selectsignal and reference wavelengths while taking into accountwavelength-related scattering. Further, in order to minimize scatteringlosses, it may be desirable to shift these measurements towardslow-scatter (low index differential) regions of the spectrum.

Another factor to optimize in wavelength selection is complex index(absorption) of the particle being measured. When using a wavelengthwhere absorption is very weak, local resonant scattering will also beweak. To increase the absorption signal, it may be preferred to use awavelength near the absorption peak of the particle. In this case, evenif there are changes in shape or orientation of the particle, theeffects on measurement signal may be weak, as there is minimalself-shading. However, if the absorption signal is very strong, not onlywill there be strong resonant scattering effects as a function ofwavelength; there is also the potential for strong orientationdependence in the measurement. This is partly as a result of scatteringdependence on orientation (not accounted for in Mie scattering models,which assume spherical particles) so that a short path through a largecross-section particle may be different than long path through smallcross-section particle. However, ignoring scattering, the pureabsorption signal of a non-spherical particle becomesorientation-dependent when absorption is high. This is a result of theexponential decay of light intensity on the path through the particlevs. the linear change with cross-sectional area. For particles with veryhigh absorption there may be a stronger mismatch as the particle rotatesrelative to the interrogating beam. Therefore, it may be desirable inmany cases to select off-peak wavelengths for absorption measurements.

FIG. 46 shows a generalized system diagram for measurement of mid-IRabsorption of a particle or cell, including but not limited to livingcells. A QCL 4602 is the light source for the system. In the context ofthe present invention, the QCL may be a mid-IR or THz-emitting quantumcascade laser, or a multiplicity of lasers. The QCLs may be fixed inwavelength or tunable with one of a number of known tuning mechanisms.In the case where a multiplicity of QCLs are used, they may be focusedonto the same measurement volume, or different volumes, and then theparticle may translate across volumes (or vice versa, with the systemtranslating across particles) and absorption/scattering from differentQCL sources measured sequentially.

As shown in FIG. 46, light 4604 emitted from the QCL 4602 is deliveredto the particle 4608 that is being measured. Light 4610 passing directlythrough the particle 4608, with some fraction absorbed according to thewavelength and the molecular composition of the particle 4608, isrelayed to an appropriate detector 4612. In an embodiment of absorptionmeasurement, where relatively uniform particle mixtures are beingmeasured, this system wherein only light 4610 that passes directlythrough the particle 4608 is measured may sufficient. However, in otherembodiments where other particles are measured (such as cells in asuspension, an emulsion, solid particles in a liquid stream, or indeedliquid droplets in air), it may be insufficient. In such embodiments,particularly where the size of particle 4608 approaches the wavelengthof light 4604, significant scattering may occur in an angle-dependentmanner, depicted here by scattered rays 4614. Unless the system isdesigned to capture or compensate for this scattered light 4614, it mayobtain misleading absorption measurements for the particle(s) 4608.

FIG. 47 shows one approach to remedying the scattering problem. QCL(s)4702 are focused using optics 4704 to form an input beam 4708 with aspecific angle, focused onto a measurement volume containing particle(s)4710. On the output side, both directly-transmitted (and shallowscatter) light 4712 as well as scattered light 4714 with a certain angleare collected using a high NA lens as part of the collection optics4718. With a sufficiently large differential between the input beammaximum angle and the output collection angle, scattering losses may bereduced. For a system with a small enough refractive index differentialbetween the particle and medium along with particles that are relativelysmall compared to the wavelength being used, this system may besufficient to remove most scattering effects from the absorptionmeasurement. As described earlier, probe wavelengths may be optimized aswell to ensure reduced scattering losses (at least to the level requiredby the accuracy required in the system).

FIG. 48 shows an alternative embodiment wherein scattered light ismeasured directly. Scattered light measurement can then be used in anumber of ways. First, it may be used to estimate the total amount ofscattered light in the system, including lost light, to correct for thisloss in the output of the system. Second, it may be used to estimateparticle parameters including index and volume (particle size), togetherwith the direct absorption measurement(s). Third, it may provide agating signal for absorption measurements, because it provides apositive signal on a zero background, while the absorption measurementmay be a small delta on a bright background.

The components of this embodiment include QCL(s) 4802 that are focusedusing input optics 4804 into an input beam 4808. Where the input beam4808 has a smaller cone angle than the output collection angle providedby collection lens 4812. The input beam 4808 is focused onto particle(s)4810 in the measurement volume. A high numerical aperture (NA)collection lens 4812 is used to collect both the transmitted light, aswell as scattered light within a given angle. Some scattered light athigh angles 4814 will be lost in the system. After the collection lens,an annular mirror 4818 is used to divert the scattered light portionthrough a focusing lens 4820 onto a detector 4822. This “scatteredlight” detector 4822 measures primarily light that is scattering fromthe particle(s). The signal from this detector 4822 may be used asdescribed above. Simultaneously, the directly-transmitted or small anglescattered light is focused by a focusing lens 4824 onto the directtransmission detector 4828 which may be the primary absorption detector.

In this embodiment, the system may operate with a multiplicity ofwavelengths to determine chemical concentrations and other particleattributes such as size. With multiple QCL wavelengths in thisembodiment, both absorption and scattering may be measured at multiplepoints on the curves as shown in example form in FIGS. 43a and 43b . Bymeasuring scattering at a known angle at multiple wavelengths, whenrefractive index varies locally with wavelength as it does in resonantmid-IR measurements, it may be possible to accurately determine particleproperties from known-angle, multi-wavelength measurements—whiledetermining specific chemical concentration as well. This is a novelcapability that is not present in conventional cytometers using visibleor near-visible light, and has not been explored in mid-IR or THzsystems because of the lack of powerful, wavelength-matched, brilliantsources. QCLs fill this gap for measurement of cells and other particlesin liquid streams.

Furthermore, the present invention may be used in a system thatdifferentiates populations of particles, such as cells, where theoutputs of the scattered light detector 4822 and direct-transmissiondetector 4828 at multiple wavelengths may be used with existingalgorithms to separate populations of particles or cells. In asorting-type system, the parameters used may be refined continuously tomaximize separability of populations. The QCL-based, absorptionresonance-tuned infrared forward transmission and scattering systemoffers multiple capabilities in a variety of applications, and may betuned accordingly.

FIG. 49 shows an embodiment that uses QCLs to measure particle size andchemical concentration through scattered light only. In this embodiment,the system works even when particles are strongly absorbing at certainresonant wavelengths (for example, when DNA is very densely packed inthe nucleus of a cell). It can also provide the advantage that allsignals are zero-based; in other words, when no particle is based,sensor readings are close to zero (even with the QCL sources poweredup). Only when particles enter the measurement volume are positivereadings present on the scatter detectors. In addition, the absolute andrelative readings on scattered light detectors 4924 at one or morewavelengths may be used to determine both particle size and chemicalcomposition (or at least concentration of one target compound within theparticle).

In FIG. 49, the components of the embodiment include two QCL sources4902 and 4910 to illustrate schematically how their signals are combinedusing collimating optics 4904 and 4912, and a beam combiner 4908 whichis optimally a dichroic thin film interference filter provided the QCLshave different wavelengths. The combined beams are focused using lens4914 onto the particle(s) 4918 in the measurement volume. Light isscattered in a wavelength- and angle-dependent manner, some of which iscaptured by collection lens 4920. A series of spatial filters 4922 (inthis case annular mirrors) are then used to select ranges of scatteringangles for detection by detectors 4924. There are a number of ways toachieve this configuration, including detectors that are segmented,blocking or reflecting filters, etc. Optionally, direct transmission ismeasured with another detector 4928. Importantly, light is onlyscattered when a particle 4918 is present in the measurement volume sothat when a particle 4918 is not present, the scattering detectors 4924detect close to zero light. In contrast, the optional detector 4928 willdetect some level of light regardless of whether a particle 4918 ispresent or not. In addition, by using more than one scattering detectorwith associated annular mirrors that sample different scattering angles,measurements of scatter dependent properties such as particle size areimproved by having an angular measure of scatter.

While this example is shown to have symmetry around the output beam (all360 degrees around an annular section is diverted into a single scatterdetector), the system may be further refined to have horizontal andvertical scatter detectors, or more detectors arranged in a circularmanner around the beam. This would allow more complex cell shapes to beaccounted for and measured, if desired. Using the scattered lightdetectors to measure scattered light at two angle ranges, and at one ormore wavelengths near absorption resonance for the target compound, onemay estimate both particle size and concentration. This may be doneeither through Mie scattering models which iteratively calculate thesequantities by achieving best fits to the data (lookup tables may begenerated in advance in high-throughput systems), or particles may beclassified empirically, either using independent measurements, or bytweaking parameters until known statistical populations are wellseparated in the output of the system. For example, in a system wheresperm cells are being separated by DNA content in order to select X- orY-carrying populations, it is known that roughly 50% of the samplecarries each. Consequently, the outputs of the detectors in the system(at different angles, wavelengths) may be weighted to achieve separationbetween two populations, without reference to a quantitative model todescribe scattering.

In a system where QCL sources are tunable, the measurement wavelengthsmay be optimized empirically as well. In the case ofapplication-specific tools, this may be done in the product developmentstage. In the case of more flexible instruments, or where the analyte(particles, and medium) may vary substantially, the sources may betunable in operation, and may either sweep through the course of ameasurement, or find an optimal measurement wavelength with a subset ofparticles and then perform the main measurement or sorting operation.The generalized function of the scattering-based system is describedhere: one or more frequencies at resonant and optionally non-resonantbands are used, and one or more angle ranges of scattered light aremeasured. The resulting signals may be used to calculate particle volumeand one or more chemical concentrations (or, in some cases, molecularconformations/configurations, which also cause changes inabsorption/refractive index profiles in the mid-IR).

The present invention may be applied to a wide range of applicationswhere infrared vibrational absorption resonances are an indicator ofinterest. One application may be in measurements of biological cells.The label-free measurement of biochemical content in cells is of stronginterest. The present invention has the potential to significantlyimprove accuracy of systems previously described by the inventor. Inaddition, it has the potential to simultaneously measure cellular orcellular component (i.e. nuclear) dimensions and chemical contents. Withembodiments of this disclosure, effective volume and concentration formultiple cellular components could be measured simultaneously usingmultiple wavelengths. For each cellular constituent (protein, DNA,lipid, etc.) a concentration and a spatial distribution figure could becalculated using resonant absorption and/or scattering signals atresonant peaks for each. For example, the invention may be applied todiagnostic applications, such as applications where a large number ofcells are measured in a high-throughput cytometry tool. Cellulardimensions are measured using scattering, and contents such as DNA, RNA,proteins, and/or metabolic products are measured using resonant IRabsorption. Cell population statistics are accumulated and outliersidentified in order to detect abnormalities to diseases. For example, atool which detects malaria from a raw blood sample could isolate DNAmeasurements from red blood cells, where DNA readings over a thresholdcould indicate presence of parasites (sizing capability of the systemused to separate readings from red blood cells from other bloodcomponents). In another example, a system based on the presentdisclosure could refine samples from circulating tumor cell (CTC)capture devices; cells may be characterized by DNA and other content aswell as size.

In another example, embodiments of the disclosure may be applied toassisted reproductive applications. A system based on the presentinvention could accurately determine the DNA content of sperm cells forhuman or animal applications, even as the nuclear volume of the cellsvaries within a patient sample. The ability to measure DNA level allowsseparation of X- and Y-bearing sperm cell for gender selectionapplications. Similar systems could screen out cells with chromosomalabnormalities, or abnormal DNA packing configurations for in vitrofertilization applications. The invention could also be applied toscoring of embryos, where chemical content and byproducts is known tocorrelate with viability, and label-free, non-ionizing radiation-basedtechniques would be highly preferable. The present invention couldcharacterize chemical content of an embryo while compensating forscattering effects, or indeed harnessing resonant scattering to producta more accurate score.

In another example, embodiments of the disclosure may be applied toregenerative medicine. The present invention may be used to refine cellpopulations by both size/shape and chemical content in order to separatepluripotent stem cells from mixtures of cells, without attaching labelsor interrogating the cells with potentially damaging radiation. Fordifferentiated cells generated from stem cells, the system may beapplied to remove any residual pluripotent stem cells before insertioninto patient tissues, in order to prevent tumor formation bynon-differentiated cells.

In another example, embodiments of the disclosure may be applied toindustrial biology. The present invention may be used to characterizeand/or sort cells for industrial processes at high speed. For example,it may be used to refine a culture of cells that produces a high numberof lipids or other products under certain conditions. Such measurementsof cellular products could be done by isolating cells in droplets whichare in turn manipulated in an oil medium as an emulsion.

In another example, embodiments of the disclosure may be applied tocontamination monitoring. Water and other substances may be monitoredfor infectious diseases without pre-processing using the presentinvention. Some filtering for particle/cell size may be performed on theinput, and then particles flowed through a measurement system. Thepresent invention's ability to characterize cell size, generalrefractive index, and resonant absorption/scattering for particularcompounds can allow it to identify specific pathogens. Where pathogensare airborne, the system may capture a sample on an open liquid surface,then flow the liquid through an analysis system. For solid samples suchas food, well-known processes for blending the sample, then filteringthem, may be used to separate potential pathogens before flowing themthrough a system based on the present invention.

Another application may be measurement of emulsions. In many cases it isuseful to measure droplets within an emulsion. This can facilitatehandling in some cases. In other cases the sample being measured isinherently an emulsion. In a droplet-based measurement system, waterdroplets suspended in oil are used to manipulate cells and discretevolumes of chemicals in a microfluidic system. The present inventioncould be used in conjunction with such systems in order to measurechemical concentrations within droplets optically, and at the same timepotentially adjust for the size of droplet. In some cases, the dropletsize measurement may be done at shorter wavelengths. In other cases, acombined measurement around mid-IR resonances is helpful to measureoverall chemical content. In some cases the analyte of interest is anemulsion of chemicals. The present invention can serve to characterizesuch mixtures both by chemical content and by particle size. This may beuseful in a number of industrial and food processes.

Another application is in the measurement of solid particles insuspension. With the present invention, it may in some cases bedesirable to build a system that places solid particles into a liquidsuspension in order to characterize them. For example a system mayinclude a capability that collects a solid sample from a surface throughscraping, milling or vacuuming to produce particles. The particles arethen introduced into a liquid medium to provide samples for measurement.Where the liquid medium may be tuned to have specific refractive indexrelationship to the particles of interest, so that it in effect becomesa “reference” in the system. The samples are then run through one ormore size filters to select particles that are appropriate formeasurement and exclude unwanted substances (including for example,size-sorting microfluidic devices such as those demonstrated by RobertAustin at Princeton University). The filtered sample containingparticles is then run through a measurement system based on the presentinvention. Even if the particles are very dense and have high opticalabsorption at resonant wavelengths, the presentscattering-compensated/enhanced measurements system may providevolumetrically-compensated chemical composition information, or bothvolume and chemical composition information. Such systems may be ofinterest in applications where diffuse reflective or ATR-typespectroscopy are currently used, but signals are insufficiently accurateor sensitive. This may include cases where there is a very thin layer ofinterest on an object (the described system serves to consolidate thelayer into a flow), or where the underlying substances interfere withmeasurement.

Emulsion-based microsystems for handling of small volumes of analyte orbiological materials, including cells, are an area with growingactivity. One common challenge in such systems is measurement of dropletor flow contents, where individual volumes are tiny. It would be highlydesirable to be able to measure the contents of such an emulsionnon-invasively, without adding additional chemicals that may disturb thechemical reaction or cellular metabolism, and without radiation thatcould disturb or harm the contents of the droplets, cells, emulsions,etc.

The unique properties that a QCL-based system brings to an emulsion ordroplet-based fluidic system include the ability to provide highspectral power density at specific wavelengths in the mid-IR and THzregime corresponding to molecular bond vibrations; and the ability tofocus this light very tightly and efficiently onto a small spot for thepurpose of interrogating individual particles, droplets, biologicalcells, etc. with micron resolution, thereby creating the ability tomeasure these at a high rate while resolving them individually.Correspondingly, the low etendue of QCL light sources allows light to bevery well and efficiently collimated in the case where it is desirableto have the angle distribution of the illuminating source to be verynarrow.

Unlike most Raman spectroscopy systems that probe the same molecularvibrations, mid-IR systems have very strong interaction with the targetmolecules. In addition, mid-IR light, including light from QCLs, has theadvantage that it is very low energy (per photon), minimizing the chanceof damaging the sample, this is particularly important when the sampleincludes biological cells. Finally, mid-IR light has the advantage oflonger wavelength vs. UV/Visible/NIR and Raman measurements; this longerwavelength reduces scattering effects and makes them more easilymanageable in a measurement system.

The fluidics may be fabricated using materials appropriate for mid-IRlight transmission and designed optically to avoid fringing andresonances at the wavelengths employed.

A number of configurations are described below. Any of theconfigurations, techniques, architectures previously described usingQCLs to interrogate particles, cells, droplets and sub-flows in fluidsare applicable to these.

One additional optical method that may be applied to the previousconfigurations disclosed herein adds polarization as a sensing modalityto the QCL-based interrogation of these particles in a flow. Ifmolecules being interrogated by mid-IR vibrational spectroscopy arearranged in specific manners within the particle being measured—forexample, DNA in helical configuration—the measured absorption at theabsorption band of the molecules will depend on the polarization of themid-IR light. One may alternately polarize the light in left and rightcircular polarization and measure the differential. The observeddifferential, so-called vibrational circular dichroism (VCD) can providea particularly sensitive measurement of chiral or helical molecules,and/or provide information about the folding or configuration of aparticular molecule within the analyzed particle/cell/droplet. QCL-VCDinterrogation of particles in a fluid may be combined with othertechniques described herein, such as scattering, or use of mid-IR activelabels/dyes, to measure certain types of target molecules.

One example of a target molecule measurement that would be a goodcandidate for QCL-VCD based measurement is DNA measurement. DNA is ahelical molecule and therefore exhibits polarization-dependentabsorption at resonant absorption bands of its constituents (phosphateand deoxyribose components on its backbone). This property may be usedto separate a DNA absorption signature from other analytes whenmeasuring using one or more QCL wavelengths. It may also be used todetermine, with high accuracy, the folding or packing state of DNAwithin a cell nucleus.

FIG. 50a shows a simple flow architecture where the fluid to be measuredflows through a channel, where it is interrogated using a mid-IR or THzQCL-derived beam (shown as a dotted ellipse in FIGS. 50a-50c .). In thecase illustrated in FIG. 50a , the transmission at one or morewavelengths through the channel is measured to determine chemicalconcentrations within the flow.

FIG. 50b shows a fluid-within-fluid flow, also within a channelstructure or manifold. At small scales, as is well known, fluids tend toremain in a laminar (non-turbulent) regime wherein mixing doesn't occur.This enables a “core” flow of analyte (shown cross hatched in the centerof FIG. 50b ) to remain centered and unmixed within a “sheath” flow, asis shown in FIG. 50b . This method of presenting an analyte (which maybe a liquid, liquid with solid particles and/or biological matter,liquid with dissolved gases, emulsion, or suspension) gives someadditional possibilities for QCL-based measurements. First, iteliminates potential artifacts arising from the laser beam (dottedellipse) crossing the edge of the fluid channel. Second, in thisconfiguration it is more straightforward to make scattering-basedmeasurements that accentuate differences between the core and sheathfluids. Refractive index (both real and imaginary) differences betweenthe sheath and core flows will result in optical interference effects(often described as Mie scattering for particles), effectively changingthe angle of some of the light transmitted through the flow. Asdiscussed earlier, in the mid-IR regime addressable by QCLs, there arerelatively narrow, resonant refractive index variations (dispersion)around absorption peaks characteristic of molecular bond vibrations.These may be exploited, with QCL-derived illumination whose angle iswell controlled, to get very sensitive concentration measurements withinthe fluid flow. Depending on the concentration of a particular moleculewithin the core flow, there will be characteristic variations inrefractive index—and therefore in observed scattering/diffractionintensity and angle—as a function of mid-IR wavelength.

The flow shown in FIG. 50b may simply be two fluids (core and sheath)flowing laminarly, but not otherwise separated. Alternatively, the flowmay be an emulsion, where the two fluids naturally remain separated. Forexample, the sheath fluid could be oil, whereas the core flow could bean aqueous solution. Several measurements could then be performed usingthe QCL-based architectures previously disclosed: the characteristics ofthe core flow (dimension, base index) could be measured at onewavelength where the molecule of interest is not resonant; then otherwavelengths could be used to measure specific chemical concentrations ofinterest, using either direct transmission/absorption measurements, orscattering-based measurements. Knowledge of the core flow diameterderived from the initial measurement may then be used to compensate thetarget-specific signal, for example accurately calculating concentrationof the chemical within the core flow. Multiple angles (directtransmission, and multiple scatter angles) may be used to calculatedimensions and concentrations.

With this as well as other architectures disclosed herein, multipleconfigurations for measuring scattering over multiple angles may beused. Discrete detectors capturing light scattered at different anglesmay be used, for example. In another configuration, a mid-IR focal planearray may be used to simultaneously measure light emerging at differentangles from the measured fluid/cell/particle. Alternatively, mirrorsystems may be used to sample portions of the output beam. Mirror arrayssuch as the Texas Instruments digital micromirrors that can be anglecontrolled may be used to sample spatial and angular portions of theemerging beam and relay them to a single detector (such as a high-speedMCT). Rotating mirrors may be used to scan the emerging beam over adetector with an aperture, either along 1 or 2 axes (useful wherepotentially asymmetric particles/droplets are being measured).

These architectures, with two or more QCL wavelengths, one or more ofwhich corresponds to wavelengths where a substance of interest has aresonant dispersion feature, may be used with multiple architecturesthat present particles, cells, fluids, droplets and the like to themid-IR beams. For example, this QCL-based resonant Mie scatteringarchitecture may be used with cells that have been mounted on a mid-IRtransparent substrate (or mid-IR reflective substrate).

FIG. 50c shows an example of a flow where the core has been broken intodroplets. These droplets may be emulsed in the sheath flow. The dropletspass through the QCL measurement beam (dotted line) and are measuredusing direct transmission and/or scattered light detection.

All of these architectures based on mid-IR QCL light may be combinedwith other measurement techniques, including optical. These include butare not limited to scattering measurements, fluorescence measurements,and others previously disclosed.

FIG. 51a shows an example of scattering efficiency (Qs) of a volume suchas a droplet, as a function of wavelength (λ). Assuming relativelyconstant refractive index for the volume as well as the surroundingmedium, scattering drops as a function of wavelength.

FIG. 51b shows scattering efficiency as a function of wavelength where achemical within the droplet has a vibrational absorption band in thewavelength range being displayed. A local increase in absorptionnecessarily corresponds (as can be determined from the Kramers-Kronigrelation) to a local resonant variation in real refractive index. Thisvariation in real index results in a local (wavelength) perturbation ofthe optical interference pattern (scattering) arising as the mid-IR beampasses through the droplet and surrounding medium. The term “droplet”here is used to mean droplet, cell, particle within another liquid,including cases where the droplet is in an emulsion.

It should be noted that several mid-IR (or other) wavelengths may beused to accurately determine chemical concentration within such asystem. The resonant dispersion (local index variation around theabsorption band) measurements are done using mid-IR QCLs. Otherwavelengths in non-resonant regimes may be used to measure the overalldroplet (size, shape, orientation) by scattering. For this, visible,NIR, or mid-IR wavelengths may be used. Visible wavelengths are alreadyused to assess size and shape in systems such as blood count toolstoday. They, however, do not have the capability of doingchemistry-specific measurements enabled by the mid-IR resonantscattering architecture disclosed herein.

FIG. 52a shows a fluidic configuration where a droplet or flow isconfined within a 2D channel or manifold, such as those found in manymicrofluidic chip architectures. Mid-IR light from one or more QCLspasses through the droplet and the surrounding medium. Depending on thewavelength and chemical concentrations within the droplet andsurrounding medium, light is absorbed and/or scattered.

FIG. 52b shows a fluidic system where droplets or a core flow iscentered within a larger, 3D core flow. This is more typical ofconventional flow cytometer cuvettes, for instance. The QCL-basedmeasurements described previously are then made of the droplet or flow,based on directly transmitted or scattered mid-IR light.

The fluidic architectures shown in cross section in FIGS. 52a-b may beused in cases where the “droplets” or “flows” are in emulsion; in otherwords, where the droplets shown here do not mix with the surroundingflow, even when stationary.

FIGS. 53a-c show a representative example of a system measuring dropletsor flows (within a sheath flow, which is not shown) using QCL-originatedmid-IR beams. They are used to illustrate scattering as a function ofthe flow/droplet content, and mid-IR wavelength, as described under FIG.53.

FIG. 53a shows the system with the sheath flow only, or a baselinestate. All mid-IR light is focused onto a single point, corresponding totraversal of the sheath fluid without angular perturbation resultingfrom refractive index differences.

FIG. 53b shows the system as a droplet or core flow are introduced. Thedroplet will cause some differential absorption of thedirectly-transmitted mid-IR light (this could be in some cases benegative, where the droplet absorbs less than the sheath flow). As afunction of refractive index and wavelength, the droplet will alsodiffract some of the mid-IR light so it exits the flow off-axis. Whenimaged onto a plane, this light will appear away from the central spot.As described earlier, this pattern will be wavelength-dependent, andaround certain resonant absorption wavelengths, will have strongerwavelength dependence. A combination of the directly-transmitted(central) signal and scattered signal as a function of mid-IR wavelengthand scattering angle (position on the plane) may then be used toestimate both droplet dimensions and specific chemical concentrationswithin the droplet. The droplet, here, could be a continuous flow ofliquid (viewed in cross-section), either in laminar flow or emulsed in asheath fluid; it could be a single droplet; it could be a biologicalcell; a solid particle; or other form of spectrally-measurable object ina liquid flow, as described earlier.

FIG. 53c shows the same droplet or flow, now with another object insideof it. For example, this could be a particle, a cell, a dense nucleus orother organelle. It could be a droplet in an emulsion containing asingle cell (or multiple cells). The system may be used again, asdescribed above, to characterize the droplet, and then to characterizethe particle within the droplet using the same techniques, often usingdifferent mid-IR wavelengths that correspond to molecular bondvibrations of interest within the contained particle. Again, this willresult in absorption of the QCL-originated light passing through thecontained particle, and scattering of the light based on refractiveindex at those wavelengths, which may show resonant dispersion aroundcharacteristic absorption lines.

In this architecture, a tunable QCL could be used to interrogate aseries of wavelengths corresponding to non-resonant (reference)wavelengths, then resonant wavelengths corresponding to the dropletfluid and particle fluid, respectively.

In some cases, the droplets may accumulate chemical species as a resultof chemical reactions that have occurred inside of them, or biologicalprocesses occurring in a cell contained within them (possiblystimulated/inhibited with additives to the droplet).

FIG. 54a shows a case where a biological cell 5410 is contained within adroplet 5420, which is contained within an emulsion. An advantage ofsuch a system, is that the droplet 5420 acts as an independent volume,not mixing with the surrounding (transport/sheath) liquid. As a result,chemical reactions can be performed at a tiny scale, and their resultsmeasured (assuming an appropriate measurement technique is used, such asthe methods described herein). Alternatively, as is shown in thisrepresentative example, a cell 5410 may be incubated in the droplet 5420with nutrients, drugs, toxins and/or other substances, in parallel withthousands or millions of other cells in a microfluidic system. At afixed time, the present invention may be used to interrogate the droplet5420 using QCL-derived light. At this point, the contents of the cell5410 may be measured directly using vibrational spectroscopy techniquesdescribed herein. However, it may also be of interest to measure thecontents of the droplets 5420, which will contain the metabolicbyproducts of the cell 5410, accumulated in the course of incubation.The byproducts and their concentration may be a powerful indicator ofcell 5410 function. The droplet 5420 contents may also be measured usingthe QCL-based techniques described herein. In some cases, only thecontents of the emulsed droplet 5420 itself will be of interest, and thetechniques described above may be used to “substract out” the opticalsignature of the cell 5410 from the transmitted and/or scattered mid-IRlight.

FIG. 54b shows a different technique, illustrated here through the useof a droplet 5430 in an emulsion. Here the droplet 5430 is formed,containing a cell 5440, but also containing a dye or label with a knownand preferably distinct mid-IR vibrational signature. As withfluorescent dyes/labels, and emerging quantum dot based tags, it isfunctionalized in order to bind to or localize in particular portions ofthe cell 5440, possibly depending on the cell 5440 phenotype, surfaceantibodies, etc.

The advantage of using a mid-IR dye/label, with readout using QCL-basedmid-IR interrogation (absorption and scattering) are multiple: (1) ahigh degree of chemical flexibility, since virtually all molecules havemid-IR fingerprints; (2) the potential for a large number of labelssimultaneously present and measured, because of the complex fingerprintsachievable; (3) well-behaved scattering at mid-IR wavelengths, at thescale of cells and organelles. Moreover, scattering, as has beendescribed, can be measured in a very chemical- and size-specific manner.As a result, it is possible to measure chemical concentration andassociated structural information using resonant Mie scattering andQCL-originated beams with well-controlled angular incidence.

In this example, the droplet 5430 is filled with a mid-IR label, whichover the course of incubation, binds to specific structures on thecontained cell 5440. By interrogating the droplet 5430 using QCL beam(s)after this incubation, one may determine how much of the label has beenconcentrated on the surface of, in the nucleus of, or within anotherorganelle of the cell 5440.

This technique of labeling using mid-IR active dyes/labels/particles maybe applied with QCL-based vibrational absorption/scatteringarchitectures previously disclosed by the inventor. It is potentially astrong complement to the label-free techniques previously disclosed, andto UV/visible/NIR techniques in broad industry use today.

FIG. 55 shows an example of the present invention used in adroplet-based system where droplets 5510 contain biological cells. Thedroplets 5510 may also be seeded with growth media, nutrients, drugs,etc. In general it may be desirable to have a set number of cells (oftenone) per droplet 5510; generally the number of cells will follow aPoisson distribution. The cells may be incubated for a fixed period atspecific conditions, and then run into a measurement volume 5550 asshown here, where microfluidic channels 5520 guide individual droplets5510 into a flow channel with a sheath flow 5530 that centers thedroplets 5540.

Alternatively, various focusing techniques, including acoustic, optical,and mechanical may be used to center the flow of droplets and space themappropriately for measurement, and in some cases, subsequent sorting.

In this configuration, multiple wavelengths of QCL (or, tunable QCL(s))may be used in conjunction with direct transmission and/or scatteringdetectors in order to determine any one or combination of the following:(1) how many cells are present within a particular droplet, potentiallyeliminating from consideration those with the wrong number of cells, orcompensating remaining measurements appropriately; (2) gatheringspectral/scattering information regarding the cells to ascertain theircontents or phenotype; (3) determine droplet volume; and (4) measurechemical contents of the droplets to measure, for example, the metabolicproducts of the cell(s) contained within. The resulting measurementscould include anything from simple characterization of what type ofcell(s) are contained within droplets, of the purpose of sorting,diagnostics or statistical measurements; what the contents of the cellsare, including contents such as DNA, RNA, protein, sugars, lipids,metabolic byproducts, etc.; what chemicals are produced by the cell. Themeasurements may be recorded, or used to sort droplets in real time.

In other configurations, the droplets may be immobilized in “pots” wherethey may be observed over time. In this manner, time series (or at leastbefore/after) measurements may be made of droplets maintained orprocessed under specific conditions. The array of pots may either betranslated across the QCL beam for one-by-one measurement, or the potsmay be unloaded sequentially past a QCL measurement point.

FIG. 56 shows another configuration where a solid sample with verysparse (potential) particles of interest is to be analyzed. The processof analyzing a bulk sample for trace chemical or biological componentsis often difficult because signal is low when the entire sample ismeasured, or highly variable if small points are measured (for example,by ATR prism using FTIR).

According to the present invention, the solid 5610 may be milled into afine powder 5620 (if it is not already in powder form), and thenintroduced into an appropriate liquid. Depending on the material beinganalyzed, this could be water, alcohol, oils or other liquids. Theparticles may then be sorted or filtered to achieve uniform size,potentially through the use of microfluidic structures. The particles5620 are then incorporated into droplets 5630 in an emulsion. Thesedroplets 5630 may, in some cases, contain not only the particles 5620but also additive chemicals or tags that attach to or react with themolecules of interest in the solid, and may produce byproductsdetectable by mid-IR vibration spectroscopy.

The droplets 5620 are then interrogated, possibly after some reactiontime, using one or more QCL beams 5640, in order to determine thecontent of the particle 5620, by direct measurement of the particle5620, by measurement of complexes formed between additives and theparticle 5620, or byproducts of reactions between the particle 5620 andadditives.

Particles here could include minerals of interest, trace pollutants orcontaminants, explosives or chemical/biological weapons traces, ormicrobes including food contaminants.

FIGS. 57a-c show flow and particle configurations that could be used inthe present invention to enhance resonant optical interferencemeasurements in the mid-IR. Here, rather than a single particle, dropletor stream producing an interference (scattering) pattern whenQCL-originated mid-IR beams hits it, multiple parallel particles orstreams are used to produce a periodic “grating” in order to enhancescattering effects and increase signal-to-noise for specific chemicalbond detection.

FIG. 57a shows parallel laminar flows 5710 which may be formed initiallyby relatively large microfluidic nozzles, and then narrowed to theappropriate dimensions, to form a liquid diffraction grating 5720 whichmay serve to measure the liquid content with very high specificity andaccuracy in the mid-IR. In such a configuration, the diffraction anglesof light imparted by the grating as light 5730 passes through it(perpendicular to the page) depends on the phase difference imparted bythe alternating fingers, and therefore on the relative refractive indexbetween the flows. As noted, where there are chemicals present in oneset of flows that have resonant absorption bands in the mid-IR, thereare associated refractive index fluctuations (resonant dispersion) nearthe same wavelengths, and two or more QCL-based measurements atwavelengths in or around these resonant features made with detectorsmeasuring off-angle transmission can result in precise chemicalmeasurements. Both resonant and non-resonant measurements may be made toaccurately determine concentration and compensate for size and operatingenvironment effects.

FIG. 57b shows a series of droplets 5740 passing through an asymmetricbeam 5750 at regular intervals to similarly form a liquid diffractiongrating.

FIG. 57c shows an architecture where particles, cells or bubbles 5760are focused using acoustic, fluidic or optical means into parallelstreams 5770 for interrogation by QCL beam(s) 5780 according to thepresent invention.

FIG. 58 shows one method by which cells or particles can be measured andsorted using QCL-based vibration spectroscopic techniques describedherein, and droplet emulsions 5890. In this example, a double emulsionis used, where cells 5885 are contained in a water-based droplet 5880,which is encased in an oil “shell” 5875 (a double, or triple emulsion),which itself is suspended in a water-based medium. According to methodsdescribed above in the invention, QCL based beam(s) 5870 are used tointerrogate the cell 5885 and/or surrounding liquid 5880 that (forexample) contains metabolic byproducts from the cell 5885. Depending onthe outcome of the measurement, the system may break open 5868 the oilshell and release the contained cell. This release step may be performedusing one of a number of means, including but not limited to: acousticforces that break up the droplet, optical pulses which force a hole intothe shell (including but not limited to mid-IR radiation which maytarget specific absorption lines for the oil), or mechanical. Afterrelease, cells 5865 still in oil cases may be separated passively byappropriate microfluidic structures from cells 5860 which have beenreleased from their shells.

In embodiments, sub-systems may be provided that may include some, butnot all, of the components of the system as a whole. For example, asub-system may include a handling system as described herein and a QCLlaser source as described here, adapted for use with a variety ofdetectors that may be provided by a third party. Similarly, a sub-systemmay be provided that includes the QCL laser source and the detector,adapted to be used with a variety of handling systems provided by thirdparties, or a sub-system may include a handling system and detector,adapted to be used with a variety of laser sources that may be providedby a different party.

Now we turn to embodiments relating to vibrational scatteringspectroscopy measurements. One architecture that may be combined withthe methods previously described is an interferometric arrangement wherea signal beam (which passes through the sample or volume of interest) isinterfered with a reference beam (which does not pass through thesample) which has been given some phase offset, in order to removebackground signal from the system.

The present disclosure is not limited to single-point detection: it maybe used in an imaging microscope configuration, where a high-contrastimage providing size, shape, density and chemical information aresimultaneously captured. Such a system may use a series of spatialoptical filters, as well as multiple or tunable mid-IR light sources,and an imaging detector (which may be a focal plane array, scanningdetector, or coded-aperture imaging system, for example) to build up animage of the sample under inspection. Such a high-contrast, resonantvibrational scattering system may be of particular interest inbiomedical applications for tissue and cell culture imaging. It shouldbe noted that a scattering-based micro-spectroscopic system such as thisone is capable of resolving sub-wavelength features within a sample, asopposed to a direct absorption spectroscopic microscope. As a result, itenables measurement of single-cell or even subcellular features,variations or changes using mid-IR spectroscopy, which allows veryspecific, label-free biochemical characterization.

Where such a microscope system based on the present invention is builtusing QCL sources, it may be desirable to include in the system one ormore measures for reducing coherence in the system, in order to avoidspeckle effects that will impact image quality. Some of these aredescribed above. They may include devices which provide time-varyingpath lengths (shorter than the integration time for the image) or longpath lengths. Some of these devices have been described by others foruse in visible-light display and microscopy systems which use lasersources.

The present invention may additionally be combined with methods by whichmultiple wavelengths may be used simultaneously to illuminate the sampleand measure scattering. Such methods may include multiple light sourceswhich are modulated in a manner such that they may be electronicallyseparated after detection; multiple light sources arranged in an array,and projected onto corresponding detector array(s); and multiple lightsources which travel through the detection volume and then are separatedusing one or several wavelength-separation techniques available(including but not limited to diffraction gratings and thin filminterference filters).

While in many cases the present invention is useful for measuring thechemical composition of particles or cells, in some applications it maybe used to measure the liquid medium itself, where the liquid medium hasa number of constituents that must be measured, for example. In suchcase, “particles” of known size or composition may be inserted into themedium, and scattering intensity as a function of angle and wavelengthmeasured and analyzed to characterize the liquid medium. Again, such ascattering-based measurement may result in significantly higher contrast(and therefore speed, accuracy) than a conventional absorptionmeasurement.

In an alternate embodiment, features are patterned into the walls of themeasurement volume such as for example, a cuvette or slow channel, wherethe features cause scattering that is then measured and analyzed tocharacterize a liquid medium. In another embodiment, scattering in aliquid medium is caused by laminar flows where the target liquid issurrounded, or alternated, with a reference liquid where the scatteringis measured and analyzed to characterize the target liquid.

FIGS. 59a-59d show two sets of refractive index and scatteringefficiency graphs corresponding to particles in a medium without andwith resonant features arising from molecular bond vibrations.

FIG. 59a shows constant refractive indices n_(p) and n_(m), respectivelyfor the particle and medium over the local wavelength (λ) range shown.This is similar to the case of cells in water in visible wavelengthranges, for example, where the constituent components of live cells haverelatively low variation in refractive index over the visible wavelengthrange.

FIG. 59b shows the scattering efficiency Q_(s) vs wavelength λ as aresult of the constant index differential shown in FIG. 59a , asdescribed by Mie scattering, which is dependent on the indexdifferential and the size of the particle. In practice, the shape of theparticle also has an impact on the scattering efficiency but this factoris left out in order to simplify this figure. At low wavelengths, theparticle scatters more light; this drops off at longer wavelengths. Inthis figure, we show two particles (P1 and P2) of the same index anddifferent size to illustrate that size change causes a differential d₁in scattering efficiency at wavelengths λ₁ and λ₂, while for a singleparticle P₁, the differential is d₂. However, there is ambiguity betweensize change, and chemical concentration change as reflected byrefractive index.

FIG. 59c shows the refractive index n vs wavelength λ of a compound thathas a local resonant absorption peak. An absorption peak causes a localfluctuation of the real refractive index of the particle n_(p). Asshown, over the same range the medium has a relatively constantrefractive index n_(m). If the particle has chemical components that donot have local resonance, they add to the baseline refractive index ofthe particle that may be observed at λ₀.

FIG. 59d shows the scattering efficiency Q_(s) vs wavelength λ of twoparticles P3 and P4. Wherein the particle P3 contains a compound with alocal resonant absorption peak as illustrated by n_(p) in FIG. 59c ,whereas P4 does not as illustrated by n_(m) in FIG. 59c . The curvesshown in FIG. 59c are offset for ease of illustration, but could becoincident except for the area where P3 displays resonant scatteringbehavior. Observation of scattering at wavelengths λ₀, λ₁ and λ₂illustrates how resonant scattering may be used within the presentdisclosure to characterize the size and chemical composition of theparticle by observing scattering efficiency at these wavelengths.Moreover, overall scattering efficiency is only one factor that may beobserved with the systems and methods described herein: intensity oflight as a function of scatter angle and wavelength may provideadditional information on particle size, chemical composition, andshape. Through the use of resonant absorption/refractive index shift andappropriate optics, far more information than is currently obtainable byvisible/near IR scattered light methods may be obtained.

The importance of this method is great: it allows measurement ormonitoring of particle sizes and chemical composition at potentiallyhigh speed, and with great accuracy, without the addition of stains orother labels commonly used in visible-light methods. The methods harnessrelatively new bright light sources in the infrared wavelength rangethat corresponds to the most chemically specific vibrational absorptionfeatures and in a low energy photon range so that sample damage duringmeasurement is reduced. Quantum cascade lasers, synchrotron sources, aswell as other mid-infrared sources may be used within the scope of theinvention.

The useful applications of the methods and systems described in thecurrent disclosure are myriad. In the non-biological area, the abilityto measure chemical makeup of small particles or droplets within amedium can provide a powerful new method for analyzing solids andliquids; by breaking them down into small particles or droplets, theymay be presented efficiently within another medium to a measurementsystem, for instance in a microfluidic flow. In this manner solids andliquids with normally very high extinction coefficients (which aretherefore difficult to measure with traditional transmissionspectroscopy; and are as a result often sampled using surface-onlytechniques such as attenuated total reflection) may be presented withsufficiently small path lengths to measure transmission spectra. Themedium may be selected to provide appropriate refractive index match (ordifferential) for the particles of interest.

FIG. 60 shows a system for providing high contrast vibrationalspectroscopy measurements of particles within a medium. This system andmethod uses relatively recent infrared light sources that provide highbrightness: synchrotron sources, and the more recent quantum cascadelasers (QCLs). A light source 6001, which may for example be one or moreQCLs, is collimated and then focused by one or more lenses 6002, 6003onto the measurement volume 6004 (here depicted as particles flowing orbeing translated by, as indicated by the arrow). The light on theincoming (left) side on the sample has a relatively low numericalaperture, compared to the capture angle on the transmitted side (right).As a result, the capturing lens 6007 captures both directly transmittedlight 6005, as well as light that has been scattered over some angle6006. A beam block 6008 is then used to block the center portion of thebeam, and therefore the light 6005 that has been transmitted directlythrough the sample. The remaining (scattered) light 6006 is focused byone or more lenses 6009, onto the detector 6010. In this manner, onlywhen a particle is in the measurement volume 6004 and scattering occurswill there be a signal on the detector 6010. Wavelengths correspondingto both non-resonant and resonant regions of the target compounds may beused on the source side, and relative scattering between wavelengthsused to determine size and chemical concentrations in the particle asherein described.

FIG. 61 shows a related system. A light source 6101 provides infraredlight that is focused by lenses 6102 and 6105 onto the measurementvolume 6106. In this embodiment, a beam block 6103 is used before theremaining infrared light 6104 is focused onto the measurement volume6106. As a result, a “hollow cone” 6107 of light is expected on theoutput side in the absence of scattering. In the case where there isscattering, some light is diffracted inward 6108 where it passes throughan aperture 6109, and is then relayed and focused by lenses 6110 and6111 to a detector 6112. In some cases, a reflective lens arrangementmay be used in place of the initial beam block, which enhances theefficiency of the system. In addition, the beam block 6103 or thesurrounding aperture may be dynamically switchable or programmable.

FIG. 62 shows a system where a collimated beam as provided by the lightsource 6201 and one or more lenses 6202, is projected onto themeasurement volume 6203. Any particle 6210 within this measurementvolume 6203 produces scattered light 6204. A lens 6205 focuses thecollimated light 6206 to a point on the Fourier plane, where a beamblock 6207 is used to block the unscattered light. The scattered light6204 is then focused by lens 6208 onto the detector 6209.

In an alternate embodiment, an infrared image sensor (not shown) isprovided at the Fourier plane to capture an image of the scattered light6204. The pixel positions in the captured image then correspond to 2Dscattering angles. This 2D scattering angle information can provideadditional information on the shape of the particles measured.

Note that each of these embodiments may also be modified to split lightinto multiple detectors according to scattering angle. This may beaccomplished through the use of elliptical annular mirrors placed in thepath of the collimated output light such as shown in FIGS. 48 and 49 forexample, each selecting a specific range of radial positions (andtherefore angles) and reflecting it to a detector.

FIG. 63 shows an architecture for simultaneously capturing scatteringangle and wavelength vs. intensity information from particles 6312 whenemploying a multiple-wavelength or broadband infrared source 6301. Here,input side optics 6302, 6303 produce a low-angle beam that is used toilluminate the measurement volume 6304, and a large numerical aperturelens 6305 is used to capture the directly-transmitted light as well asscattered light. A slit 6306 is then used to sample a portion 6307 ofthe resulting beam. The slit 6306 may be positioned to include thedirectly-transmitted light (to measure absorption), or shifted in orderto sample only scattered light, and therefore allow use of a detectorarray with lower dynamic range, if needed. A wavelength dispersiondevice 6308 (depicted here as a reflective grating) is then used todisperse the slit-transmitted portion 6309 of the light onto an infraredimage sensor or a 2D detector array 6310. The result is that one axisalong the 2D array 6310 corresponds to wavelength (λ, shown as the xaxis in the figure), and the other axis corresponds totransmission/scattering angle (θ_(s), shown as the y axis in the figure)from the particle(s) 6312 in the measurement volume 6304. In this case,the broadband infrared light source 6301 may be (but is not limited to)a broadband IR synchrotron source, multiple QCLs that are multiplexedinto a single beam, or a Fabry-Perot type QCL with broadband emission.

Note that all of these methods and systems may additionally havereference detectors incorporated in order to measure infrared sourceoutput and normalize measurements. This may be accomplished, forexample, through the use of a beam splitter (not shown) placed in thecollimated beam (before the sample) that reflects some portion of theemitted light to a reference detector (not shown). The output of thisdetector is then used to normalize readings from the signal detector inthe system. This may compensate for noise or variation in the IR sourceor its drive electronics.

FIG. 69 shows an example embodiment employing programmable spatial lightmodulators to control angle of illumination and detection in an infraredmicrospectroscopy system. This system may be an imaging microscope, forexample, where contrast is enhanced and chemical-specific scattering ismeasured through the use of resonant scattering techniques describedherein. Programmable spatial light modulators 6904 and 6908 may be usedon the input path (before sample), the output path (after it has passedthrough the sample), or in some cases, both input and output.

In this simplified example, a light source 6901, for example one or moreQCL sources (potentially tunable), is collimated using optics 6902. Forimaging applications, a decoherence module 6903 is used to providediversity in phases over time on the measurement volume 6906 includingthe sample particle or cell—this can be done using a number of methods,some of which are described herein—for example by moving diffractiongratings in the path of the beam. Subsequently, a spatial lightmodulator (SLM) 6904 is used to selectively transmit the collimatedbeam. This allows the measurement volume 6906 to be illuminated fromspecific angles, or combinations of angles, for example to achievehigher contrast through the use of darkfield techniques. The SLM 6904may employ one of a number of known SLM technologies, including but notlimited to fixed apertures or masks that are switched in or out of thesystem; Texas Instruments' digital micromirror array; or liquid-crystalbased technologies. The SLMs may be either transmissive orreflective-type arrays. For simplicity, a transmissive array SLM 6904 isshown here. SLMs may modulate amplitude, phase, or both aspects of theincoming beam. The light is then focused onto the measurement volume6906 using optics 6905. After passing through the sample, light(containing both directly-transmitted as well as scattered light) isrecollimated by optics 6907 and then transmitted into another SLM 6908.This SLM has the effect of limiting which angles from the sample areultimately transmitted to the detector 6910. Finally, focusing optics6909 focus the light onto a detector 6910. In the case of a microscopysystem, this detector may be an infrared image sensor or a 2-dimensionalfocal plane array (FPA). Alternatively, where an FPA is too expensive ordoes not match performance requirements, a scanning detector systemcould be used, or another SLM could be employed to successively sampleportions of the image and relay them onto a single detector. In anotherembodiment, this architecture may be combined with optics that separatelight by wavelength (for example, a diffraction grating) forsimultaneous measurement.

The present disclosure may be used as the basis for a microscope thatdelivers high contrast measurement of structure as well as chemicalcontent based on both absorption and scattering characteristics. By useof scattering, and in particular resonant scattering from vibrationalfeatures of molecules or biomolecules, the microscope may obtain imagesthat elucidate high-resolution chemical and structural aspects of asample, including sub-wavelength features. For example, such amicroscope may be used to obtain images that indicate the size anddensity of subcellular structures (organelles) with specific biochemicalmakeup.

In another embodiment, the present invention may be used together with asample holder such as a well plate, that is used to measure a series ofdistinct biological samples; in each measurement characterizing mid-IRabsorption and/or scattering at one or more wavelengths, and possiblymaking an imaging measurement of these cells. The measurements may berepeated over time to assess changes in the cells, including binding ofbiomolecules, cytotoxicity, cell proliferation, cell morphology, celladhesion, and cell death. The present invention may be used to measuremany or all of the interactions and changes that other opticalbiosensors measure (for example the biosensor disclosed in U.S. Pat. No.7,300,803, Label-free methods for performing assays using a colorimetricresonant reflectance optical biosensor, Lin et al—incorporated herein byreference in its entirety). Moreover, unlike traditional optical sensorssuch as those described in U.S. Pat. No. 7,300,803, the presentinvention allows biochemical-specific measurement of the targets throughthe use of wavelengths corresponding to molecular bond vibrations, asopposed to visible or near infrared wavelengths where biomolecules havegenerally constant refractive indices and absorption vs. the medium andsubstrate materials. This enables architectures for measuring effects oncells with high specificity and high throughput.

Now we turn to a set of embodiments relating to particle displacementmeasurement. In a further embodiment, the present invention utilizesinfrared light in ranges where molecules have specific absorption bands,to separate absorption signals from particles and surrounding/permeatingmedium, allowing measurement of displacement of the medium, and therebynon-medium particle volume.

The method works as follows: one or more infrared wavelengths where themedium is absorptive, and particles under inspection do not haveabsorption bands, is transmitted through a known (or constant) thickness(or path length) of medium containing the particles. In one embodiment,the volume may be a flow channel filled with the medium through whichparticles pass. In another embodiment, the medium can be a solid in theform of a sheet, film or strip of constant thickness containingparticles. As the particles pass through the thickness, they displace avolume of medium, causing a reduction in the light absorbed at themedium-specific wavelength. The reduction in absorption when theparticle is present is used to calculate the non-medium content of theparticle. This and related embodiments may be used where single-particleinformation is desired in order to generate population statistics, oreven to sort particles by non-medium content.

In another embodiment, a known thickness may be filled with the mediumand one or more particles (an emulsion, for example); the transmissionat the medium-specific wavelength is measured to determine the totaldisplacement of medium by particles within the volume. Where particlesizes are well known, this method may be used to measure concentrationof particles in the medium. For example, in biological applications,this method may be used to rapidly assess cell concentration in asample, without occlusion and other effects that plague other methods.An example of such an application would be a rapid sperm cell count inreproductive applications. This measurement could be supplementedthrough the use of other markers (for example, DNA-specific absorption).

The aforementioned embodiments describe the use of absorptionmeasurements in transmission, to measure medium displacement byparticles. Several techniques described herein may be used to enhancethe contrast of the signal generated using the current method. Forexample, an interferometric optical arrangement may be used to reducethe background signal when only medium is present in the measurementvolume; in this case, a fractionally much larger signal is observed whena particle displaces the medium. In another embodiment, a scatteringsignal is used to enhance contrast. In this case, one or morewavelengths are selected where the real refractive index of the mediumvaries significantly from the particle, and the amount of lightscattered by the particle is measured. In the preferred embodiment, atleast two wavelengths are used in a region where the real refractiveindex for the medium varies sharply (i.e. where there is a strongabsorption band for the medium), and the index of the particles measuredis relatively constant (i.e. where there are no strong absorption bandsfor the particles). Scattered light at these two or more wavelengths ismeasured; together these measurements are used to calculate thenon-medium displacement of the particle.

The present invention may be combined with a variety of other methodsfor measuring particles, including but not limited to visible-lightscattering measurements (to enhance accuracy of these measurements, forexample), fluorescence techniques, and spectroscopy based techniquessuch as Raman or infrared spectroscopy. For example, the present methodmay be used to measure total non-medium volume of a biological cell inorder to adjust measurements made by mid-IR absorption and/or scatteringtechniques described herein, and thereby arrive at more accuratemeasurement of a biological cell's biochemical makeup.

The present invention may be applied to a variety of problems whereparticle volume and more specifically displacement must be measuredaccurately. Examples include measurement of solids in a liquid medium,measurement of liquid within liquid (emulsions, for example), andbiological cells within a liquid medium.

In some cases it may be useful to use a relatively long wavelengthcompared to the size of the particles being measured. Since mid-IR lighthas a longer wavelength than visible or NIR, there is relatively littlescattering produced by particles that are on the order of a micron insize or less, and therefore relatively little size and shape-dependentscattering occurs. As a result, a straightforward absorption measurementmay be done using mid-IR light, with the light that is scattered atsmall angles included in the transmission measurement.

In some cases where the present invention is used in conjunction withother spectroscopic measurements, the calculated displacement of themedium may be used to compensate other spectral measurements for theeffect of absorption by the medium in the wavelength bands where thetarget analyte is measured.

FIG. 68 illustrates a method by which non-water content of particles orcells may be measured with high accuracy using infrared techniquesdescribed herein.

In the mid-IR, water absorbs uniformly over the range, and in someplaces has very strong, broad absorption peaks. As a result, when aparticle or droplet passes through the measurement volume within a watermedium, there is some displacement of water, and a drop in absorption atthese wavelengths. This drop in absorption may be used to measure thenon-water volume of a particle or droplet very precisely by directtransmission measurement. This technique could be used anywhere wherewater absorbs strongly (and preferably, the target particle has nostrong vibrational absorption features). However, a transmission methodfavors the selection of long wavelengths relative to the particle sizein order to minimize losses from scattered but uncaptured light, whichcould cause measurement errors. As a result, for many biologicalapplications, it is desirable to use mid-IR wavelengths where waterabsorption is strong and wavelengths are on the order of the size of thetarget (particle, cell or nucleus). For other applications, for examplewhere target particles are smaller, this method could utilize shorterwavelengths (1-2 microns, short wavelength infrared).

This method may complement other measurements made using vibrationalabsorption techniques described herein, or with more conventionalcytometry techniques (scattering or fluorescence) to measure totalamount of non-water content in cells or other particles (or droplets).

Rather than direct transmission measurement, this method may be builtusing the systems described herein to obtain particle measurements byscattered light techniques. For example, one of the strong waterabsorption peaks may be selected; around this peak, there is significantvariation of its real refractive index (according to the Kramers-Kronigrelationship, and as described elsewhere in this text). The lightscattered by the particle, as a function of wavelength about thisresonant water peak, may be measured in order to ascertain waterdisplacement (non-water content) of the particle. This method may applyequally to non-water media, or where solutions are used (and thereforethe displacement of the solute is measured).

FIG. 68 shows the absorption spectrum (absorption vs wavenumber) for anexample medium, phosphate-buffered saline (PBS), and dried cells withthe spectra scaled for better illustration. While in some casesabsorption peaks coincide between the PBS curve and the DRY CELLs curve,there are regions dominated by water absorption as shown in the PBScurve—for example, at under 900 cm-1 wavenumbers, but also local smallerpeaks such as the ones at 2100 cm-1 and 3600 cm-1. A third absorptionspectrum curve is shown for cells in PBS (denoted as PBS+CELLS). As canbe seen, in some portions of the PBS+CELLS curve, cell-specificabsorption peaks may be seen (for protein, DNA, lipids, etc.), however,in areas where there is strong water absorption, the presence of thecells reduces the absorption of the PBS+CELLS curve compared to the PBScurve. This indicates displacement of water by non-water components ofthe cells being measured.

In some cases, the water displacement measured by this technique may beused to correct other spectral readings (for example, to get moreaccurate content information based on other absorption peaks). Forexample, the apparent “negative absorbance” at water peaks 6801 or 6802may be used to calculate the amount of water displaced by cells in theexample shown here. This in turn may be used to compensate themeasurement of cellular content at points such as 6803.

Now we turn to a set of embodiments related to interferometric particlespectroscopy. As described previously, the ability to measure particlessuspended in liquid, either individually or in aggregate, using mid-IRspectroscopic methods (including the use of QCL sources) has significantimplications in a number of applications, both in the biomedical marketand in other markets. The methods and systems described herein provide abasis for high accuracy, high speed, label-free measurements ofparticles or cells that were not previously possible.

The use of new sources such as the mid-IR QCL provide high intensity,spectral purity and the ability to efficiently focus light, and as aresult may remove light source intensity as a limiting factor, evenwhere a liquid medium may absorb a significant portion of the mid-IRradiation. Thus the combination of QCLs with liquid-medium based systemsprovides new and unique measurement capabilities, including but notlimited to measurement of live or undamaged biological cells within aliquid medium (flowing in a channel, or in a well plate array or otherfluidic matrix, for example).

Even with the interposing liquid of thickness/path length sufficient forhandling and sustaining cells/particles, QCLs provide sufficient signalfor detection in reasonable periods of time, unlike traditional mid-IRsources such as globars. With a very intense light source such as a QCL,the limiting factor of such a system may now be the saturation limit ofthe corresponding mid-IR detectors(s). In many cases, the amount oflight absorbed by a passing particle will only be a very small fractionof the total light transmitted through the measurement volume, becauseconcentrations may be low, absorption coefficients may be low, and oftenbecause the size of the particle(s) will be small compared to the beamcross-section in the measurement volume (or, where multiple particlesare measured, the concentration of particles is relatively low in thedetection volume). For example, in a flow type system, it is generallypreferable to use a large (and potentially asymmetric) beamcross-section in order to minimize position dependence of signal asparticles pass through the beam.

Moreover, when operating detectors with a high baseline intensity (anddeviations corresponding to signal), noise becomes dominated by shotnoise, which rises as the square root of intensity. Therefore, if thesignal is a fixed percentage of the baseline, four times the intensityyields (theoretically, excluding other noise sources) only twice thesignal-to-noise ratio.

Ideally, one could reduce the baseline level of power on the detector toa very low level, and observe the same absolute signal corresponding toa passing particle. This would significantly enhance signal-to-noiseratio, and eliminate detector saturation as an issue. Several methodsfor reducing the impact of high baseline have already been described.One is straightforward—the use of AC-coupled preamplificationelectronics with the mid-IR detector; however, while this conditions theoutput of the detector to optimize subsequent processing and/ordigitization, it does not address the issues of signal-to-noise orsaturation. Another method that has been described at length is the useof scattered light rather than directly-transmitted light. Since in manymid-IR particle measurements, the majority of light will be transmittedin a straight path without angular scattering, if only scattered lightis measured, the measured intensity will be very low when no particle ispresent (assuming no other scattering features in the system), and willincrease as particles pass. As has been described herein, the intensityof scattered light as a function of wavelength and scatter angle may beused to determine multiple particle characteristics including volume,density, shape and chemical composition (through appropriate selectionof wavelengths close to resonant vibrational bands of target compounds).

However, it would be advantageous to have a method by which intensity onthe detector in such a system could be zero-based (or much closer tozero-based) which did not rely inherently on scattering, sincescattering is dependent on a number of factors, and may not besufficiently strong in some cases to uniquely determine particlecharacteristics (though, ideally, it could function equally well insystems where scattering is measured as well).

In yet another embodiment of the present invention, a method and systemis provided that utilizes one or more mid-IR sources to probe particlesin liquid, together with interferometric optical arrangements that allowelimination of a significant portion of background intensity on one ormore mid-IR detectors. The invention enables higher accuracy and/orhigher speed measurement of particles in mid-IR spectroscopic systems.

A basic implementation of the present invention is constructed asfollows: a QCL source (or sources) is used to generate mid-IR light atspecific wavelength(s); the light is collimated and then split into twoarms: a signal arm, and a reference arm; the signal arm is passedthrough the measurement volume where it may be absorbed/scattered by oneor more particles, when they are present; the reference arm, on theother hand is treated by the use of a phase-delay device in order to setits delay compared to the reference arm; in addition, the reference arm(or signal arm) may be passed through an attenuator in order to matchthe intensities between the signal and reference arms in the base state;the arms are then recombined with another beam splitter/combiner andrelayed to a mid-IR detector. In this way, the presence of particles inthe signal beam causes it to be imbalanced relative to the referencebeam so that when the signal beam and reference beam are combined, thesignal produced by the particles is enhanced. The phase delay in thereference arm is controlled to create a delay between wavefronts thateffectively cancels much of the observed intensity when no sample ispresent in the measurement volume, by imposing roughly a half-wave delayon the reference arm relative to the signal arm. An optimal delay may befound in order to achieve a combination of low baseline and sufficientsignal in response to a particle (or particles) in the measurementvolume. An attenuator (which may be variable) is used to matchintensities of the reference and signal arms to further lower thebaseline intensity observed by the detector. In this way, the presenceof particles in the signal beam causes it to be imbalanced relative tothe reference beam so that when the signal beam and reference beam arecombined, the wavefronts are not fully cancelled and a high contrastsignal is produced that is associated with the particles.

As a result of this method, the ratio of the incremental signal inresponse to a particle to the baseline intensity may be significantlyincreased. Correspondingly, more accurate, faster measurements may beachieved. The system may be built in a number of configurations, allemploying the same basic method for enhancing signal-to-baseline.

Another embodiment splits the optical signal after the measurementvolume; the arms are then focused onto two different spatial filters.The “signal” arm travels through a spatial filter that admits low aswell as high frequencies. The “reference” arm travels through a spatialfilter that admits only low frequencies, and therefore corresponds tothe “DC” background of light transmitted through the measurement volume.Again, a phase delay is imposed on the reference arm, so that thereference and signal arms largely cancel each other when no particle ispresent in the volume. When a particle (or particles) is present, itssignal is transmitted by the all-pass spatial filter on the signal arm,but not by the low-pass filter (a pinhole aperture) on the referencearm, causing a differential in intensity between the arms, and aresulting incomplete cancellation (and therefore an observed signal) atthe detector.

In another embodiment, both signal and reference arms travel through themeasurement volume, but offset from one another. In this manner, when aparticle is present in one of the beams, a differential signal isobserved. In one embodiment, these arms may be in the form of opticalwaveguides passing through a liquid channel or volume (in this case,signal and reference splitting, recombining, and phase delay mayindividually or in combination be implemented in a waveguide structure).

Splitting and combining of signal and reference paths may beaccomplished through a number of means, including but not limited tothin film beamsplitters and diffraction gratings. In addition,polarization techniques may be used to split opposite polarizationcomponents, relay them at an offset through the measurement volume, andthen recombine them; thus any differential causes a shift of thepolarization from the baseline, which may be detected through the use ofa polarization analyzer and mid-IR detector (this technique is known as“differential interference contrast”).

The present invention may be applied both to single-channel systems(where detectors are non-imaging) as well as imaging systems in themid-IR spectroscopic range. For example, a microscopy system employing atunable QCL source, the interferometric arrangement described in thepresent invention, and mid-IR imaging means (including but not limitedto mid-IR focal plane arrays, scanning detectors, and detectorsoutfitted with spatial light modulators in order to build up imagesthrough multiple exposures) may be combined for the purpose of imagingsamples comprising one or more particles. These particles may be in aliquid medium; further they may be biological particles including butnot limited to live biological cells, tissue samples, bacteria, bloodsamples, or other liquids.

The present invention may be combined with other means of enhancingsignal from particle samples. These include but are not limited to useof scattering-based measurements that use intensity as a function ofscattering angle and wavelength as a means to characterize a sample. Inthis case, identical spatial filtering/handling may be used in each pathin order to illuminate the sample with a specific range(s) of angles,and to capture only specific range(s) of angles transmitted through thesample.

For example, one or more QCL sources may be first collimated, thenpassed through a spatial filter (which ultimately sets the angles ofillumination of the sample); then split into sample and reference beamsas described above; after passing through the sample (on the signal arm)and phase delay (reference side) and recollimated, the beams may berecombined, and then passed through another spatial filter, before beingultimately focused on a mid-IR detector. The present invention appliesto multiple-wavelength systems as well. In many cases wavelengths usedto interrogate particles are relatively closely spaced, and as such mayuse the same reference arm phase shift, albeit with slightly non-optimalcancellation of baseline on the detector. Where highest signal-to-noiseratio or contrast is required, phase may be changed for eachinterrogating wavelength in turn; alternatively, material with acompensating amount of dispersion may be added to the reference path.

FIG. 64 shows a method for enhancing contrast in an infrared vibrationalspectral particle measurement system. This method can be used withtransmitted light, scattered light, or a combination thereof. It is aninterferometer-based system with a signal arm and reference arm, whichallows at least partial cancellation of background in the system. Theinfrared light source 6401, for example a QCL, is collimated by optics6402, and then split using a beamsplitter 6403 into two arms, the signalarm (straight) and reference arm (downward). The signal arm light 6404is focused by optics 6405 onto the measurement volume 6406 (shown here,for example, as a flow with particles in it). The transmitted light iscaptured and collimated by optics 6407, then recombined with the lightthat has passed through the reference arm using another beamsplitter6408, and then focused by one or more lenses 6409 onto a detector 6410(note the light passing through the beam combiner 6408 may then berelayed to and focused onto a reference detector (not shown), whichtracks the level of light coming through the reference arm; this may beused to adjust for variations in mid-IR source power over time). Thelight in the reference arm is sent through a variable delay block 6412(an example construction of delay block 6412 is shown in FIG. 64a ), bymirrors 6411 and 6413, that allows the path length to be modified at asub-wavelength increment. This enables the phase of the light recombinedwith the signal arm to be precisely tuned to cancel the background inthe signal arm, and provide high contrast when a sample appears in themeasurement volume. In addition, the reference or signal arm should inmost cases include a variable attenuator (not shown) in order to allowadjustment for a precise amplitude match between signal and referencebeams. Finally, an additional focusing section with a pinhole aperture(not shown) may be used before the detector 6410 in order to simplifyalignment between signal and reference arms and ensure “clean” beams areinterfered when finally focused on the detector 6410. This interferencedesign may be combined with the other methods described here,specifically with methods to provide contrast based on scattered light.

FIG. 64a shows an example of a phase delay system or delay block 6412that may be used in an interferometric vibrational spectroscopy systemsuch as the one shown in FIG. 64. The reference beam 6414 is reflectedby a fixed mirror 6415 onto a moveable mirror assembly 6416 whichcarries mirrors at right angles 6417, and which may be translated(vertically as shown) in order to set a precise path length for thereference beam, and therefore delay compared to the signal beam. Thelight is returned to another fixed mirror 6418 and to the output beam6419 which is recombined with the signal arm of the system to produce aninterferometric signal. The path length through the delay block 6412 istypically adjusted to significantly reduce baseline signal, and providemaximum fractional signal when a particle enters the measurement volume6406.

Now we turn to a set of embodiments relating to spatially-enhancedparticle spectroscopy. In a further embodiment, an additional method,with multiple potential system embodiments, is provided utilizingpatterned mid-IR light in the measurement volume in order to improvesignal-to-noise ratio. The use of spatial patterning of illuminationwithin the volume allows a characteristic pattern to be generated as aparticle passes through the volume (or, as the beam is scanned over asample) to increase visibility of the signal, ultimately increasingaccuracy and/or detection speed.

In this embodiment of this system, multiple distinct sources arearranged optically such that they are focused into distinct spots in themeasurement volume. This requires careful alignment of optics in orderto align these spots properly in the volume, if the sources are indeedseparate (for example, individually packaged DFB-QCLs or tunable QCLs).Another version of this embodiment using monolithic arrays of QCLs tocreate the illumination pattern may be quite simple, as the QCL arraycan simply be re-imaged onto the measurement volume.

Another embodiment of this system projects multiple “images” of eachsource onto the measurement volume. For example, through the use of adiffraction or phase grating, it is possible to create a series of spotsfrom a single QCL source. When using a diffraction grating, multipleorders of diffracted light may be imaged as a series of spots or linesin the measurement volume. Then, as the particle or cell passes throughthe measurement volume, the periodic illumination results in acharacteristic temporal pattern on the mid-IR detector(s) used in thesystem.

This spatial pattern may be 1-, 2- or even 3-dimensional depending onthe volume being interrogated. For example, where a linear flow is beinginterrogated, a 1-dimensional pattern along the axis of flow may beoptimal. Where a 2D volume such as a sample between IR microscopeslides, or within a well plate array, is being measured, a 2D patternmay be optimal; finally in a volumetric measurement or thicker samplesuch as a tissue sample, a 3D pattern may be required.

While diffraction gratings or even slit arrays may be sufficient togenerate simple patterns, this method is not limited to these. Patternedphase gratings (reflective or transmissive) or even devices that providedynamic phase shaping (such as beam shaping mirrors) may be used toprovide arbitrary illumination pattern in the detection volume.

Where there are multiple wavelengths, each wavelength may be processedby a separate grating or phase modulator, and therefore be projectedonto the detection volume with a distinct pattern; then the beams fromthese independently-modulated wavelengths are projected onto the volume.With such a configuration, a single detector may be used, and componentsfrom multiple wavelengths distinguished from one another as a particlepasses through the combined illumination field by the characteristicpatterns of each wavelength, with relative absorption and/or scatteringat these wavelengths calculated. In addition, this configuration mayallow the trajectory of the particle through the illumination field tobe estimated, in order to further refine the spectroscopic datacollected. In this embodiment, each wavelength may be projected with adifferent spatial frequency and/or respective angle in order to makethem easily separable in the electronic domain. They may be projectedonto the measurement volume in non-periodic patterns as well, includingin optimized pseudo-random patterns.

In another embodiment, multiple wavelengths may be combined into asingle beam, and then processed together by a diffractive element. Wherein this case, each wavelength is diffracted at a different angle fromthe element. The results of this angular variation vs. wavelength, afterthe light is focused onto the measurement volume, is to produce a“spread” of illumination in the measurement volume according towavelength. In the case of discrete sources that are combined, thisallows a series of illuminated regions to be formed. Again, this allowsfor more efficient, higher SNR separation of signals from the backgroundafter detection.

In another embodiment, one or more sources with broadband emission, suchas Fabry-Perot QCLs, are used with a diffraction grating. In this case,the emission from this source is spread across the measurement volumeaccording to wavelength. As a particle passes over one or more of thesegraded regions, the electronic pattern produced using the mid-IRdetector indicates absorption or scattering as a function of wavelength,allowing chemical composition (and/or size, shape, etc.) to be computed.

In all of these embodiments, after passing through the measurementvolume, the mid-IR light may be re-combined into a single collimatedbeam through the used of a complementary diffractive element, if focusonto a single small detector is required. Where the mid-IR detector issufficiently large, this element may be skipped. Alternatively, thespatial pattern projected onto the measurement volume may be imaged ontoan array of detectors for individual detection. The present inventionmay be combined with other methods described herein, including but notlimited to resonant scatter-based systems, as well as systems based oninterferometry that enhance signal contrast and signal-to-noise ratio.

FIG. 65 shows a method for forming multiple spots 6506 within themeasurement volume 6507 for the purpose of enhancing the signal from aparticle passing through the measurement volume 6507. For example, in aflow system, having a series of evenly-spaced spots of light may providean electronic signature that is easier to separate from background andnoise by using analog or digital processing. Some examples of potentialspot patterns are shown in FIG. 67a-c . The infrared beam from one ormore sources 6501 is collimated using optics 6502 and then a diffractiveelement 6503 is used (here shown as transmissive, but may be reflective)to diffract light into multiple orders 6504, which are focused onto themeasurement volume 6507 using input side optics 6505. Particles or cellspassing through the measurement volume 6507 pass through one or more ofthese multiple orders 6504 and associated spots. Transmitted light iscollected and recollimated by output side optics 6508, and then anotherdiffractive element 6509 is used to recombine the orders into a singlebeam that is focused by optics 6510 onto the detector 6511. Additionalelements (not shown) are possible to destructively cancel the backgroundsignal prior to the detector.

In an alternate embodiment, the second diffractive element 6509 isomitted, and the light is focused onto an infrared image sensor or adetector array whose elements (pixels) correspond to the array of spotsprojected onto the measurement volume. Thus, each detector will have ahigher-contrast signal as the particle passes through the correspondingspot in the measurement volume. This method may be combined with othermethods disclosed herein; including methods of increasing contrast basedon (resonant and/or non-resonant) scattering characteristics, andinterference methods for increasing signal contrast.

FIG. 66 shows a configuration where an IR source 6601 with multiplewavelengths (for example, a FP QCL with broadband emission) is used toprobe particles in a medium in measurement volume 6610. In thisconfiguration the light is collimated by optics 6602, then a grating6603 is used to diffract different wavelengths at different angles oflight 6604 and multiple orders of light, which are focused by lens 6605to provide for example, diffracted light 6607 (shown here passingthrough a spatial filter 6606 to only allow first order diffracted lightto pass into the measurement volume 6610). The diffracted light 6607 isthen focused, by lenses 6608 and 6609, onto the measurement volume 6610.As a result of the diffractive element 6603, a graded continuum or agraded series of spots 6611 in order of wavelength is projected onto themeasurement volume 6610. For example, a FP QCL, which typically emits arange of discrete wavelengths, may be used to form a series of spots(corresponding to FP cavity modes) along the axis of a liquid flowcarrying particles to be interrogated.

As a result, a particle or cell passing through the measurement volume6610 will in succession be interrogated by a series (or continuum) ofwavelengths. Depending on its size and composition, the particle or cellwill exhibit specific absorption and scattering properties as a functionof wavelength, causing changes in light transmitted either directly oroff-angle through the measurement volume. The system measures this timeseries of absorption/scattering signals and processes the data in orderto characterize the particle or cell in terms of chemical composition,size and/or shape.

In this figure one of several possible configurations on the output sideis shown. Where the transmitted beams are focused by one or more lenses6612 and recombined into a single beam through the use of anotherdiffractive element 6613 (shown here as a transmissive grating which isone of several possible devices), and focused by lens 6614 onto a singledetector 6615. As a particle (or particles) sweep through a detectionvolume, the detector picks up changes in order to build a completespectral “waveform” of the particle.

Alternatively, the wavelength recombination element (here shown asdiffractive element 6613) may be left out, and an array of detectors(not shown) may be used instead to detect individual wavelengthsseparately, increasing signal-to-background. This method may be combinedwith one of the scattering-based measurements described earlier, orspatial filtering may be applied as described earlier to separate lightthat is scattered within one or more ranges of angles and this light canthen be used to detect scattering as a function of wavelength as theparticle passes through the measurement volume.

Specially designed “coded” spatial apertures with a designed field ofopen regions may be used to generate very specific, characteristicresponses based on a particle's absorption and scatteringcharacteristics as a function of wavelength. Such apertures may bestatic (for example, thin metal with holes laser-cut or etched into itto provide transmissive regions) or programmable (for example, arrays ofmirrors which either relay a portion of the beam to a detector, or sendit to a beam dump). With the use of such a programmable spatial filter,the system may be optimized for a particular application (for example,characterizing cells by phenotype or chemical content) at production, atthe start of a run in a laboratory, or even in real time as a runprogresses (optimize continuously for maximum separation of two or morepopulations of cells, for example).

For such configurations, it may be desirable to use a beam that isasymmetric, such that it forms a “line” across the measurement volume(and where there are multiple wavelengths, they form a series ofparallel lines such as shown in FIGS. 67b and 67c ). The first functionof this shaping is to reduce lateral position dependence as a particlepasses through the measurement volume. The second is to provide a beamthat is easy to separate by wavelength along one axis (short axis, whichcorresponds to the axis along which wavelengths are distributed), andeasy to separate by scattering angle along the other (long axis) whichhas comparatively low numerical aperture and therefore a small angle ofwavelengths entering the sample.

For all of these methods as applied to particle measurements, inaddition to newer IR sources including synchrotron IR sources and QCLsources, more traditional IR sources such as globars, but also CO₂ lasersources may be incorporated as part of the system.

FIG. 67a-c shows several potential configurations for focused light on ameasurement volume where there are multiple spots being formed such asin the system shown in FIG. 65 or other. These spots may be formedthrough the use of a diffractive element, or through the use of multipleIR sources that are arranged such that they project onto different spots(examples include QCL arrays, or QCLs whose collimated beams arecombined slightly off-angle). Although shown here as discrete spots, thespots may be more diffuse so that they represent a continuum, forexample where a continuous broadband source is wavelength-dispersedalong the vertical axis.

The most simple configuration shown in FIG. 67a simply symmetricallyrelays the beams to a series of circular or close-to-circular spots. Theadvantage of this configuration is that it maximizes signal as aparticle passes through one of the spots. One disadvantage of the simpleconfiguration shown in FIG. 67a is that the signal from the particlepassing through the spots is highly dependent on its lateral position(left to right in this diagram). If it is off-center, the signals areshorter and less intense than when it is on-center. Where the spotsrepresent a series of wavelengths, it may be possible to compensate bylooking at relative signal from one wavelength to the next (assuming itpasses through the same portion of each beam).

A more stable spot configuration for most applications is shown in FIG.67b . In this case, asymmetric lenses (cylindrical lenses, for example)are used to focus the beam strongly along one axis, and with a lowernumerical aperture along the other. This results in elliptical spots asshown in FIGS. 67b and 67c . While the power density may be lower inthis configuration than in the simple spots shown in FIG. 67a , theadvantage of this design is that it is relatively insensitive to smallvariations in lateral (left-right) position of particles as they passthrough the measurement volume. A second advantage of this design, wherescattering angle is of interest, is that the small angle of the beamhorizontally allows for better separation of light scattered at an angleby the particle.

For example, if the spots along the vertical axis here represent aseries of wavelengths, a particle would pass through the series ofspots, and scatter a certain portion of each; optical (spatial)filtering to block directly-transmitted light may then by applied to thetransmitted/scattered light, and wavelengths recombined and focused ontoa single detector. For each particle passing through the volume then, aseries of signals corresponding to scatter at each wavelength isgenerated on the detector. Given the absolute and relative intensitiesof these signals, the system may characterize the particle by size andchemical composition (through both resonant and non-resonant scatteringeffects).

An alternative configuration is shown in FIG. 67c . Here the path ofparticles through the interrogation volume is purposely angled such thatthe signal from the system clearly indicates the lateral position of theparticle as it passes through the volume. Note that many patterns forinterrogation are possible. Indeed, even 2D spot arrays (or a series ofspots run in sequence, using a programmable spatial filter) may be usedto resolve position of a particle, even while using a single detector.

FIG. 70 shows an example in which two wavelengths are projected onto themeasurement volume (in this example, the particles translate along thex-axis relative to the projected light); each wavelength has beentreated with a diffractive phase element in order to form a distinctseries of spots 7010 and 7020 along the axis 7040 of particleinterrogation. As the particle 7030 moves through the series of spots7010 and 7020 for the two respective wavelengths, the particle 7030absorbs and/or scatters each wavelength differently; the signal(s) aremeasured on one or more mid-IR detectors. The pattern imposed on eachwavelength causes a characteristic signal pattern, which may beseparated in order to measure relative absorption and/or scattering ateach of the wavelengths.

In a further embodiment, the measurement methods described herein can beused to determine the molecular structure of a chemical constituent of aparticle or cell. In addition, the measure methods can be used to purifysuspensions of particles or cells in a liquid medium wherein particlesor cells with different contained molecular structures are present.Further, the measurement methods can be used in association with aprocess that changes the constituent molecular structure of particles orcells in a manufacturing system for making particles or cells with aspecific molecular structure.

In yet another embodiment, the measurement methods described herein canbe used to determine the crystal structure of a chemical constituent ofa particle or cell. In addition, the measure methods can be used topurify suspensions of particles or cells in a liquid medium whereinparticles or cells with different contained crystal structures arepresent. Further, the measurement methods can be used in associationwith a process that changes the constituent crystal structure ofparticles or cells in a manufacturing system for making particles orcells with a specific crystal structure.

While only a few embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that manychanges and modifications may be made thereunto without departing fromthe spirit and scope of the present invention as described in thefollowing claims. All patent applications and patents, both foreign anddomestic, and all other publications referenced herein are incorporatedherein in their entireties to the full extent permitted by law.

The methods and systems described herein may be deployed in part or inwhole through a machine that executes computer software, program codes,and/or instructions on a processor. The present invention may beimplemented as a method on the machine, as a system or apparatus as partof or in relation to the machine, or as a computer program productembodied in a computer readable medium executing on one or more of themachines. In embodiments, the processor may be part of a server, cloudserver, client, network infrastructure, mobile computing platform,stationary computing platform, or other computing platform. A processormay be any kind of computational or processing device capable ofexecuting program instructions, codes, binary instructions and the like.The processor may be or may include a signal processor, digitalprocessor, embedded processor, microprocessor or any variant such as aco-processor (math co-processor, graphic co-processor, communicationco-processor and the like) and the like that may directly or indirectlyfacilitate execution of program code or program instructions storedthereon. In addition, the processor may enable execution of multipleprograms, threads, and codes. The threads may be executed simultaneouslyto enhance the performance of the processor and to facilitatesimultaneous operations of the application. By way of implementation,methods, program codes, program instructions and the like describedherein may be implemented in one or more thread. The thread may spawnother threads that may have assigned priorities associated with them;the processor may execute these threads based on priority or any otherorder based on instructions provided in the program code. The processor,or any machine utilizing one, may include memory that stores methods,codes, instructions and programs as described herein and elsewhere. Theprocessor may access a storage medium through an interface that maystore methods, codes, and instructions as described herein andelsewhere. The storage medium associated with the processor for storingmethods, programs, codes, program instructions or other type ofinstructions capable of being executed by the computing or processingdevice may include but may not be limited to one or more of a CD-ROM,DVD, memory, hard disk, flash drive, RAM, ROM, cache and the like.

A processor may include one or more cores that may enhance speed andperformance of a multiprocessor. In embodiments, the process may be adual core processor, quad core processors, other chip-levelmultiprocessor and the like that combine two or more independent cores(called a die).

The methods and systems described herein may be deployed in part or inwhole through a machine that executes computer software on a server,client, firewall, gateway, hub, router, or other such computer and/ornetworking hardware. The software program may be associated with aserver that may include a file server, print server, domain server,internet server, intranet server, cloud server, and other variants suchas secondary server, host server, distributed server and the like. Theserver may include one or more of memories, processors, computerreadable media, storage media, ports (physical and virtual),communication devices, and interfaces capable of accessing otherservers, clients, machines, and devices through a wired or a wirelessmedium, and the like. The methods, programs, or codes as describedherein and elsewhere may be executed by the server. In addition, otherdevices required for execution of methods as described in thisapplication may be considered as a part of the infrastructure associatedwith the server.

The server may provide an interface to other devices including, withoutlimitation, clients, other servers, printers, database servers, printservers, file servers, communication servers, distributed servers,social networks, and the like. Additionally, this coupling and/orconnection may facilitate remote execution of program across thenetwork. The networking of some or all of these devices may facilitateparallel processing of a program or method at one or more locationwithout deviating from the scope of the disclosure. In addition, any ofthe devices attached to the server through an interface may include atleast one storage medium capable of storing methods, programs, codeand/or instructions. A central repository may provide programinstructions to be executed on different devices. In thisimplementation, the remote repository may act as a storage medium forprogram code, instructions, and programs.

The software program may be associated with a client that may include afile client, print client, domain client, internet client, intranetclient and other variants such as secondary client, host client,distributed client and the like. The client may include one or more ofmemories, processors, computer readable media, storage media, ports(physical and virtual), communication devices, and interfaces capable ofaccessing other clients, servers, machines, and devices through a wiredor a wireless medium, and the like. The methods, programs, or codes asdescribed herein and elsewhere may be executed by the client. Inaddition, other devices required for execution of methods as describedin this application may be considered as a part of the infrastructureassociated with the client.

The client may provide an interface to other devices including, withoutlimitation, servers, other clients, printers, database servers, printservers, file servers, communication servers, distributed servers andthe like. Additionally, this coupling and/or connection may facilitateremote execution of program across the network. The networking of someor all of these devices may facilitate parallel processing of a programor method at one or more location without deviating from the scope ofthe disclosure. In addition, any of the devices attached to the clientthrough an interface may include at least one storage medium capable ofstoring methods, programs, applications, code and/or instructions. Acentral repository may provide program instructions to be executed ondifferent devices. In this implementation, the remote repository may actas a storage medium for program code, instructions, and programs.

The methods and systems described herein may be deployed in part or inwhole through network infrastructures. The network infrastructure mayinclude elements such as computing devices, servers, routers, hubs,firewalls, clients, personal computers, communication devices, routingdevices and other active and passive devices, modules and/or componentsas known in the art. The computing and/or non-computing device(s)associated with the network infrastructure may include, apart from othercomponents, a storage medium such as flash memory, buffer, stack, RAM,ROM and the like. The processes, methods, program codes, instructionsdescribed herein and elsewhere may be executed by one or more of thenetwork infrastructural elements. The methods and systems describedherein may be adapted for use with any kind of private, community, orhybrid cloud computing network or cloud computing environment, includingthose which involve features of software as a service (SaaS), platformas a service (PaaS), and/or infrastructure as a service (IaaS).

The methods, program codes, and instructions described herein andelsewhere may be implemented on a cellular network having multiplecells. The cellular network may either be frequency division multipleaccess (FDMA) network or code division multiple access (CDMA) network.The cellular network may include mobile devices, cell sites, basestations, repeaters, antennas, towers, and the like. The cell networkmay be a GSM, GPRS, 3G, EVDO, mesh, or other networks types.

The methods, program codes, and instructions described herein andelsewhere may be implemented on or through mobile devices. The mobiledevices may include navigation devices, cell phones, mobile phones,mobile personal digital assistants, laptops, palmtops, netbooks, pagers,electronic books readers, music players and the like. These devices mayinclude, apart from other components, a storage medium such as a flashmemory, buffer, RAM, ROM and one or more computing devices. Thecomputing devices associated with mobile devices may be enabled toexecute program codes, methods, and instructions stored thereon.Alternatively, the mobile devices may be configured to executeinstructions in collaboration with other devices. The mobile devices maycommunicate with base stations interfaced with servers and configured toexecute program codes. The mobile devices may communicate on apeer-to-peer network, mesh network, or other communications network. Theprogram code may be stored on the storage medium associated with theserver and executed by a computing device embedded within the server.The base station may include a computing device and a storage medium.The storage device may store program codes and instructions executed bythe computing devices associated with the base station.

The computer software, program codes, and/or instructions may be storedand/or accessed on machine readable media that may include: computercomponents, devices, and recording media that retain digital data usedfor computing for some interval of time; semiconductor storage known asrandom access memory (RAM); mass storage typically for more permanentstorage, such as optical discs, forms of magnetic storage like harddisks, tapes, drums, cards and other types; processor registers, cachememory, volatile memory, non-volatile memory; optical storage such asCD, DVD; removable media such as flash memory (e.g. USB sticks or keys),floppy disks, magnetic tape, paper tape, punch cards, standalone RAMdisks, Zip drives, removable mass storage, off-line, and the like; othercomputer memory such as dynamic memory, static memory, read/writestorage, mutable storage, read only, random access, sequential access,location addressable, file addressable, content addressable, networkattached storage, storage area network, bar codes, magnetic ink, and thelike.

The methods and systems described herein may transform physical and/oror intangible items from one state to another. The methods and systemsdescribed herein may also transform data representing physical and/orintangible items from one state to another.

The elements described and depicted herein, including in flow charts andblock diagrams throughout the figures, imply logical boundaries betweenthe elements. However, according to software or hardware engineeringpractices, the depicted elements and the functions thereof may beimplemented on machines through computer executable media having aprocessor capable of executing program instructions stored thereon as amonolithic software structure, as standalone software modules, or asmodules that employ external routines, code, services, and so forth, orany combination of these, and all such implementations may be within thescope of the present disclosure. Examples of such machines may include,but may not be limited to, personal digital assistants, laptops,personal computers, mobile phones, other handheld computing devices,medical equipment, wired or wireless communication devices, transducers,chips, calculators, satellites, tablet PCs, electronic books, gadgets,electronic devices, devices having artificial intelligence, computingdevices, networking equipment, servers, routers and the like.Furthermore, the elements depicted in the flow chart and block diagramsor any other logical component may be implemented on a machine capableof executing program instructions. Thus, while the foregoing drawingsand descriptions set forth functional aspects of the disclosed systems,no particular arrangement of software for implementing these functionalaspects should be inferred from these descriptions unless explicitlystated or otherwise clear from the context. Similarly, it will beappreciated that the various steps identified and described above may bevaried, and that the order of steps may be adapted to particularapplications of the techniques disclosed herein. All such variations andmodifications are intended to fall within the scope of this disclosure.As such, the depiction and/or description of an order for various stepsshould not be understood to require a particular order of execution forthose steps, unless required by a particular application, or explicitlystated or otherwise clear from the context.

The methods and/or processes described above, and steps associatedtherewith, may be realized in hardware, software or any combination ofhardware and software suitable for a particular application. Thehardware may include a general-purpose computer and/or dedicatedcomputing device or specific computing device or particular aspect orcomponent of a specific computing device. The processes may be realizedin one or more microprocessors, microcontrollers, embeddedmicrocontrollers, programmable digital signal processors or otherprogrammable device, along with internal and/or external memory. Theprocesses may also, or instead, be embodied in an application specificintegrated circuit, a programmable gate array, programmable array logic,or any other device or combination of devices that may be configured toprocess electronic signals. It will further be appreciated that one ormore of the processes may be realized as a computer executable codecapable of being executed on a machine-readable medium.

The computer executable code may be created using a structuredprogramming language such as C, an object oriented programming languagesuch as C++, or any other high-level or low-level programming language(including assembly languages, hardware description languages, anddatabase programming languages and technologies) that may be stored,compiled or interpreted to run on one of the above devices, as well asheterogeneous combinations of processors, processor architectures, orcombinations of different hardware and software, or any other machinecapable of executing program instructions.

Thus, in one aspect, methods described above and combinations thereofmay be embodied in computer executable code that, when executing on oneor more computing devices, performs the steps thereof. In anotheraspect, the methods may be embodied in systems that perform the stepsthereof, and may be distributed across devices in a number of ways, orall of the functionality may be integrated into a dedicated, standalonedevice or other hardware. In another aspect, the means for performingthe steps associated with the processes described above may include anyof the hardware and/or software described above. All such permutationsand combinations are intended to fall within the scope of the presentdisclosure.

While the disclosure has been disclosed in connection with the preferredembodiments shown and described in detail, various modifications andimprovements thereon will become readily apparent to those skilled inthe art. Accordingly, the spirit and scope of the present disclosure isnot to be limited by the foregoing examples, but is to be understood inthe broadest sense allowable by law.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) is to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the disclosureand does not pose a limitation on the scope of the disclosure unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe disclosure.

While the foregoing written description enables one of ordinary skill tomake and use what is considered presently to be the best mode thereof,those of ordinary skill will understand and appreciate the existence ofvariations, combinations, and equivalents of the specific embodiment,method, and examples herein. The disclosure should therefore not belimited by the above described embodiment, method, and examples, but byall embodiments and methods within the scope and spirit of thedisclosure.

All documents referenced herein are hereby incorporated by reference.

What is claimed is:
 1. A method for providing spectroscopic measurementsof particles with favorable signal to noise, the method comprising:providing a mid-infrared laser light source that emits coherentmid-infrared light; splitting the emitted mid-infrared light into twobeams; introducing a sample comprising a medium with at least oneparticle to a measurement volume; directing at least one of the twobeams through the measurement volume and the at least one particle;directing at least one of the other of the two beams through themeasurement volume without the at least one particle; recombining thetwo beams into a recombined beam after the at least one beam has passedthrough the measurement volume, wherein prior to recombination, one ofthe two beams represents the medium and particle and the other of thetwo beams represents the medium, and wherein a phase delay apparatus isused to create a relative phase delay between the two beams such thatthe two beams destructively interfere in the recombined beam when noparticle is present in the measurement volume; measuring the recombinedbeam with at least one mid-infrared detector; detecting a change in atleast one of the angle and intensity in the recombined beam due to theat least one particle; and determining a property of the at least oneparticle based on the detected change.
 2. The method of claim 1, whereinthe property is at least one of a chemical composition, a physicalcharacteristic, a size, a shape, a refractive index, a density, a DNAcontent, a protein content, a lipid content, a sugar content, an RNAcontent, and a chemical makeup.
 3. The method of claim 1, wherein the atleast one particle is at least one of a biological cell, a tissuesample, a bacterium, a blood sample, and an embryo.
 4. The method ofclaim 1, wherein the medium is a liquid and the at least one particle isan emulsion.
 5. The method of claim 1, further comprising; passing atleast one of the beams through an attenuator to decrease the intensityof that beam.
 6. The method of claim 1, wherein at least one of thebeams passes through a low-pass or high-pass spatial filter beforerecombining.
 7. The method of claim 1, wherein the at least one particleis a single particle.
 8. The method of claim 1, wherein the laser lightsource is a quantum cascade laser.
 9. A method for providingspectroscopic measurements of particles with favorable signal to noiseratio, comprising: providing a mid-infrared laser light source thatemits a coherent mid-infrared light beam; splitting the light beam intotwo beams; presenting a sample comprising a medium with particles to ameasurement volume, wherein a single particle is present in themeasurement volume at a time; directing at least one of the two beamsthrough both the measurement volume and the single particle; directingat least one of the other of the two beams through the measurementvolume without the single particle; recombining the two beams into arecombined beam after the two beams have passed through the measurementvolume, wherein prior to recombination, one of the two beams representsthe medium and the particle and the other of the two beams representsthe medium, wherein a phase delay apparatus is used to create a relativephase delay between the two beams such that the two beams destructivelyinterfere in the recombined beam when no particle is present in themeasurement volume; measuring the recombined beam with a mid-infrareddetector; detecting a change at the mid-infrared detector resulting fromat least one of resonant mid-infrared absorption, non-resonantmid-infrared absorption, and scattering by the particle in themeasurement volume; and determining a property of the particle based onthe detected change.
 10. The method of claim 1, wherein the laser is aquantum cascade laser.
 11. The method of claim 9, wherein the particleis at least one of a biological cell, a tissue sample, a bacterium, ablood sample, and an embryo.
 12. The method of claim 9, wherein themedium is a liquid and the particle is an emulsion.
 13. The method ofclaim 9, wherein the property comprises at least one of a chemicalcomposition, a physical characteristic, a size, a shape, a refractiveindex, a density, a DNA content, a protein content, a lipid content, asugar content, an RNA content, and a chemical makeup.
 14. Aspectroscopic apparatus with favorable signal to noise ratio fordetermining characteristics of particles in a medium, the apparatuscomprising: a mid-infrared light source providing one or morewavelengths in a coherent light beam; a measurement volume configured tocontain a sample to be measured, the sample comprising a medium withparticles, wherein a single particle is present in the measurementvolume at a time; a first beam splitter that, when active, splits thelight beam into at least two beams; optics that are configured to directat least one of the at least two beams through the measurement volumeand through the single particle in the measurement volume direct atleast one of the other of the two beams through the measurement volumewithout the single particle; a phase delay apparatus on one of the atleast two beams that is configured to result in destructive interferencebetween the at least two beams when no particle is present in themeasurement volume; a beam combiner that is configured to recombine theat least two beams to provide a recombined beam after the at least oneof the at least two beams has passed through the measurement volume,wherein prior to recombination, one of the at least two beams representsthe medium and particle and the other of the at least two beamsrepresents the medium, wherein the phase delay apparatus is used tocreate a relative phase delay between the at least two beams such thatthe at least two beams destructively interfere in the recombined beamwhen no particle is present in the measurement volume; a detector thatis configured to detect changes in transmitted or scattered light due tothe single particle in the measurement volume in the recombined beam;and a processor that determines a property of the particle in themeasurement volume based on the detected changes.
 15. The apparatus ofclaim 14, wherein the light source is at least one of a laser and asynchrotron.
 16. The apparatus of claim 15, wherein the laser is aquantum cascade laser.
 17. The apparatus of claim 14, wherein thedetector is at least one of a mid-infrared focal plane array, amid-infrared image sensor, a scanning detector, and a detector with aspatial light modulator.
 18. The apparatus of claim 14, wherein thedetector measures intensity as a function of scattering angle andwavelength.
 19. The apparatus of claim 14, further comprising: anattenuator configured such that one of the at least two beams is able tobe passed through it to change the intensity of that beam.
 20. Theapparatus of claim 14, further comprising: a low-pass or high-passspatial filter, both configured such that at least one of the beams isable to be passed through the filter before the beams are recombined.21. The apparatus of claim 14, wherein the phase delay apparatusincludes an adjustable mirror assembly.